Forebrain-specific ablation of phospholipase Cγ1 causes manic-like behavior.

Manic episodes are one of the major diagnostic symptoms in a spectrum of neuropsychiatric disorders that include schizophrenia, obsessive-compulsive disorder and bipolar disorder (BD). Despite a possible association between BD and the gene encoding phospholipase Cγ1 (PLCG1), its etiological basis remains unclear. Here, we report that mice lacking phospholipase Cγ1 (PLCγ1) in the forebrain (Plcg1f/f; CaMKII) exhibit hyperactivity, decreased anxiety-like behavior, reduced depressive-related behavior, hyperhedonia, hyperphagia, impaired learning and memory and exaggerated startle responses. Inhibitory transmission in hippocampal pyramidal neurons and striatal dopamine receptor D1-expressing neurons of Plcg1-deficient mice was significantly reduced. The decrease in inhibitory transmission is likely due to a reduced number of γ-aminobutyric acid (GABA)-ergic boutons, which may result from impaired localization and/or stabilization of postsynaptic CaMKII (Ca2+/calmodulin-dependent protein kinase II) at inhibitory synapses. Moreover, mutant mice display impaired brain-derived neurotrophic factor-tropomyosin receptor kinase B-dependent synaptic plasticity in the hippocampus, which could account for deficits of spatial memory. Lithium and valproate, the drugs presently used to treat mania associated with BD, rescued the hyperactive phenotypes of Plcg1f/f; CaMKII mice. These findings provide evidence that PLCγ1 is critical for synaptic function and plasticity and that the loss of PLCγ1 from the forebrain results in manic-like behavior.

Immunosuppressive therapy versus alternative donor hematopoietic stem cell transplantation for children with severe aplastic anemia who lack an HLA-matched familial donor.

We compared the outcomes of immunosuppressive treatment (IST) with those of alternative donor hematopoietic stem cell transplantation (HSCT) in children and adolescents with severe aplastic anemia (SAA). The medical records of 42 patients with SAA who received frontline IST (N=19) or frontline HSCT with an alternative donor (N=23) between 1998 and 2012 were analyzed retrospectively. Six patients responded in the frontline IST group, whereas 11 underwent salvage HSCT after IST failure. Twenty-one of 23 patients who underwent frontline HSCT survived without treatment failure. The estimated failure-free survival rate of the frontline HSCT group was higher than that of the frontline IST group (91.3% vs 30.7% respectively, P<0.001). Six of 11 patients who underwent salvage HSCT experienced event-free survival (EFS). The estimated EFS of the frontline HSCT group was higher than that of the salvage HSCT group (91.3% vs 50.9% respectively, P=0.015). The outcome of alternative donor HSCT was better than commonly reported rates, especially in patients who underwent frontline HSCT. These results suggest that frontline alternative donor HSCT may be a better treatment option than IST for children and adolescents with SAA who lack a human leukocyte Ag-matched familial donor.

Post-maturation zona perforation improves porcine parthenogenetic trophoblast culture.

This study was designed to optimize a method to improve porcine parthenogenetic embryo hatching and trophoblast culture. Mature oocytes (D0PPA) and day 6 blastocysts (D6PPA) were perforated with a 20 µm diameter needle for assisted hatching. The two groups showed a significant difference in hatching rate and blastocyst cell doubling when compared to a non-perforated control group. D0PPA blastocysts were able to form tertiary trophoblast colonies but D6PPA and control groups were not able to grow beyond primary colonies. Quantitative real-time PCR analysis showed significant differences in BAX, BAX/BCL2L1 and HSP70-2 mRNA expression between the experimental groups.

Cancer cells promote survival through depletion of the von Hippel-Lindau tumor suppressor by protein crosslinking.

Nuclear factor-κB (NF-κB) and insulin-like growth factor-1 (IGF-1)-mediated signaling is associated with different tumors including renal cell carcinoma. NF-κB- and IGF-1-mediated signaling is found to be inhibited in the presence of wild-type von Hippel-Lindau (VHL) tumor suppresser gene. Therefore, negative regulator of VHL may be a good target for regulating NF-κB and IGF-1R. In this study, we found that VHL, a tumor suppressor protein that downregulates the NF-κB activity and the stability of IGF-1R was depleted by TGase 2 through polymerization via crosslinking and proteasomal degradation in kidney, breast and ovary cancer cell lines. We also found that TGase 2 knockdown promotes hypoxia-inducible factor 1α (HIF-1α) degradation, and thereby decrease HIF-1α transcriptional activity. Importantly, VHL expression was decreased in vivo in TGase-2-transgenic mice, and this was associated with increased NF-κB activity and the levels of expression of IGF-1R, HIF-1α and erythropoietin in kidney tissue. These results suggest a novel mechanism of regulation of the VHL tumor suppressor by TGase 2 that appears to be independent of the known cancer regulatory mechanisms.

The ASPIRE approach for TIA risk stratification.

BACKGROUND: The risk of stroke after transient ischemic attack (TIA) is elevated in the days to weeks after TIA. A variety of prediction rules to predict stroke risk have been suggested. In Alberta a triage algorithm to facilitate urgent access based on risk level was agreed upon for the province. Patients with ABCD2 score ≥ 4, or motor or speech symptoms lasting greater than five minutes, or with atrial fibrillation were considered high risk (the ASPIRE approach). We assessed the ability of the ASPIRE approach to identify patients at risk for stroke. METHODS: We retrospectively reviewed charts from 573 consecutive patients diagnosed with TIA in Foothills Hospital emergency room from 2002 through 2005. We recorded clinical and event details and identified the risk of stroke at three months. RESULTS: Among 573 patients the 90-day risk of stroke was 4.7% (95% CI 3.0%, 6.4%). 78% of the patients were identified as high risk using this approach. In patients defined as high risk on the ASPIRE approach there was a 6.3% (95% CI 4.2%, 8.9%) risk of stroke. In patients defined as low risk using the ASPIRE approach there were no recurrent strokes (100% negative predictive value). In contrast, two patients with low ABCD2 scores (ABCD2 score < 4) suffered recurrent strokes. CONCLUSION: The ASPIRE approach has a perfect negative predictive value in the population in predicting stroke. However, this high sensitivity comes at a cost of identifying most patients as high risk.

Laparoscopic gatrojejunostomy for palliation of gastric outlet obstruction in unresectable gastric cancer.

BACKGROUND: Gastric bypass through laparotomy is required traditionally when gastric outlet obstruction occurs secondary to a disease process (e.g., unresectable cancer). The recent trend toward minimally invasive procedures has led us to apply laparoscopic bypass surgery for gastric obstruction caused by unresectable advanced gastric cancer. METHODS: From March 1998 to February 2000, 78 gastrojejunostomies (GJ) (45 open [OGJ] and 33 laparoscopic [LGJ] procedures) were performed for palliation of gastric outlet obstruction caused by advanced gastric, duodenal, papilla of vater, and pancreatic cancers at the Asan Medical Center. In 68 patients with advanced gastric cancer, OGJ (n = 38) and LGJ (n = 30) were performed. Of these, 10 OGJ patients were compared with 10 diagnosis-matched LGJ control subjects who underwent surgery during the same period in terms of age, gender, American Society of Anesthesiology (ASA) grading, previous abdominal surgery, operating time, time to oral food intake, pain-killer consumption, postoperative hospital stay, immune response, morbidity, and mortality. Immune parameters including serum white blood cells (WBC) count, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), cortisol, and erythrocyte sedimentation rate (ESR) levels were assessed preoperatively and on postoperative days 1 and 3 between the two groups. With the patients under the general endotracheal anesthesia, we applied an upper midline incision in OGJ and inserted four trocars in LGJ. Side-to-side gastrojejunostomy was performed in a standard fashion. In LGJ, intracorporeal suture using 2-0 vicryl was performed to repair the gastrotomy and jejunotomy site after gastrojejunostomy using a 30-mm or 45-mm Endo-GIA stapler. RESULTS: There were no significant differences between OGJ and LGJ in terms of gender, age, ASA grading, and previous abdominal surgery. In OGJ, antecolic isoperistaltic GJ was performed in 10 cases, but 8 antecolic and 2 retrocolic approaches were performed in LGJ with no conversion to open surgery. Operating time (113.5 +/- 11.2 vs 100.5 +/- 9.8 min), pain-killer consumption (540 +/- 123.2 vs 430 +/- 58.2 mg), and postoperative hospital stay (12.5 +/- 3.9 vs 8.5 +/- 2.9 days) were reported, respectively. Serum WBC and cortisol levels were slightly increased in both groups preoperatively and on postoperative days 1 and 3. Serum ESR, TNF-alpha, and IL-6 levels were significantly increased in the OGJ patients. Postoperative complications (9 with OGJ and 2 with LGJ) and postoperative death (1 in each group) occurred. During the follow-up period (3-23 months), there was one case of readmission in each group because of anemia and generalized pain. CONCLUSIONS: Laparoscopic GJ for the palliation of unresectable advanced gastric cancer can achieve excellent results with less suppression of immune function, lower morbidity, greater improvement of hemodynamic activities, and earlier recovery of bowel movements than OGJ.

Adaptor protein Lad relays PDGF signal to Grb2 in lung cells: a tissue-specific PDGF signal transduction.

Lad was previously identified as an adaptor protein binding to the SH2 domain of Lck (1). Specific detection of Lad mRNA in lung cells, as well as, in T cells led us to investigate the signaling pathways regulating Lad in lung cells. We found that (i) upon PDGF stimulation, Lad expression is induced in lung cells, especially in the bronchial epithelial cells; (ii) Lad is tyrosine phosphorylated upon PDGF stimulation and is associated with PDGF receptor; (iii) upon PDGF stimulation, Grb2 is recruited to Lad in human embryonic lung cells; (iv) overexpression of Lad elevated AP-1 promoter activity by two- to threefold, whereas dominant negative Lad abrogated PDGF-dependent activation of AP-1 promoter. These results provide a novel mechanism of PDGF-dependent signaling, in which Lad acts as an adaptor in a tissue-specific manner, linking PDGF signal to Grb2 and subsequent activation of AP-1.

Redox modulation of the NMDA receptor.

Redox modulation has been recognized to be an important mechanism of regulation for the N-methyl-D-aspartate (NMDA) receptor. Sulfhydryl reducing agents enhance, whereas oxidizing agents decrease, NMDA-evoked currents. Multiple cysteine residues located in different NMDA receptor subunits have been identified as molecular determinants underlying redox modulation. The NMDA receptor is also regulated by nitric oxide (NO)-related species directly, not involving cyclic GMP, but the molecular mechanism of this action has heretofore not been entirely clear. The confusion arose at least partly due to the fact that various redox forms of NO (NO+, NO*, NO-, each having an additional electron compared with the previous) have distinct mechanisms of action. Recently, a critical cysteine residue (Cys 399) on the NR2A subunit has been shown to react under physiological conditions with NO by S-nitrosylation (transfer of the NO+ to cysteine thiol) or by reaction with NO- (nitroxyl anion) to underlie this form of modulation.

Mutation analysis of Korean patients with citrullinemia.

Citrullinemia is an autosomal recessive disease due to the mutations in the argininosuccinate synthetase (ASS) gene. Mutation analysis was performed on three Korean patients with citrullinemia. All of the three patients had the splicing mutation previously reported as IVS6-2A>G mutation. Two had Gly324Ser mutation and the other patient had a 67-bp insertion mutation in exon 15. The IVS6-2A>G mutation was reported to be found frequently in Japanese patients with citrullinemia, but Caucasian patients showed the extreme mutational heterogeneity. Although a limited number of Korean patients were studied, the IVS6-2A>G mutation appears to be one of the most frequent mutant alleles in Korean patients with citrullinemia. The Gly324Ser mutation identified in two patients also suggests the possible high frequency of this mutation in Korean patients as well.

Laparoscopy in the management of gastric submucosal tumors.

BACKGROUND: Gastric tumors, including early gastric cancers, can be safely removed laparoscopically. They do not require an open laparotomy. METHODS: From March 1995 to December 1998, we used laparoscopy to resect gastric submucosal lesions in 32 patients. There were 22 men and 10 women. The patients ranged in age from 23 to 67 years (median, 51.4 yr). The lesions were located in the upper third in one patient, in the middle third in 20 patients, and in the lower third in 11 patients. The tumors ranged in size from 2 to 6 cm in diameter. The operative procedures were wedge resection in 19 patients, wedge resection with gastrotomy in two patients, intragastric surgery in nine patients, intragastric surgery with gastrotomy in one patient, and proximal gastrectomy in one patient, using a four- or five-port technique. The exophytic mass was resected with an Endo-GIA, and the tumors on the mucosal surface were exposed via a gastrotomy and excised. The gastrotomy was closed with an intracorporeal suture. In all cases, the operation was finished after the confirmation of tumor-free margins on frozen-section biopsy specimens. RESULTS: The duration of the operation ranged from 80 to 180 mins. The final pathologic findings were leiomyoma in 24 patients, adenomyoma in three patients, hyperplastic polyp in two patients, lipoma in one patient, hamartoma in one patient, and leiomyosarcoma in one patient. One case (3.1%) was converted to a mini-laparotomy due to technical difficulty; in one other case, more margin was resected laparoscopically due to the tumor-positive margin; and in one further patient, leakage was repaired by laparoscopic suturing on the 1st postoperative day. There were no other major complications and no deaths. The hospital stay ranged from 6 to 7 days. The maximum follow-up to date in these patients, including a case of leiomyosarcoma, was 42 months. There has been no evidence of tumor recurrence. CONCLUSION: The application of laparoscopy to submucosal tumors of the stomach is technically feasible, safe, and useful. It should be considered a viable alternative to open surgery and gastroscopic management because of its low invasiveness and good postoperative results.

Direct interaction of the CD38 cytoplasmic tail and the Lck SH2 domain. Cd38 transduces T cell activation signals through associated Lck.

CD38 ligation has been shown to induce activation of intracellular signaling cascade in T lymphocytes through a Lck-dependent pathway. However, it is not clear how Lck initiates the CD38-mediated signaling process. In the present study, we showed that CD38 and Lck were physically associated through the cytoplasmic tail and the Src homology 2 domain, respectively. This was evidenced by coimmunoprecipitation of Lck with CD38 and Lck with isolated CD38 cytoplasmic domain from T cell lysate, cell lysate of COS-7 cells cotransfected with cDNAs of Lck and CD38, or a mixture of in vitro translated CD38 and Lck. Because the CD38 cytoplasmic domain does not contain any tyrosine residue, the interaction should be independent of phosphotyrosine. The interaction was further confirmed by in vitro interaction between a purified Lck Src homology 2 domain and a nonphosphosynthetic peptide corresponding to the membrane proximal region of the CD38 cytoplasmic domain. In addition, CD38 ligation resulted in an elevated tyrosine kinase activity of the CD38-associated Lck and ultimate activation of interleukin-2 gene transcription. Furthermore, expression of a kinase-deficient Lck mutant suppressed interleukin-2 gene activation in a dose-dependent manner. These results strongly suggested that CD38 ligation indeed tranduced signals for T cell activation using its associated Lck.

Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.

Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

Er-Al-codoped silicate planar light waveguide-type amplifier fabricated by radio-frequency sputtering.

A planar light waveguide-type optical amplifier was designed and fabricated. A core layer of Er-Al-codoped SiO>(2) glass was deposited onto a silica cladding layer by rf sputtering. In the Er-doped core layer the average Er and Al concentrations were 0.77 and 11.37 wt.%, respectively. We achieved 5 dB of gain with 20 mW of 980-nm pump power by using -20 dBm of 1546-nm input signal power.

Attenuation of NMDA receptor activity and neurotoxicity by nitroxyl anion, NO-.

Recent evidence indicates that the NO-related species, nitroxyl anion (NO), is produced in physiological systems by several redox metal-containing proteins, including hemoglobin, nitric oxide synthase (NOS), superoxide dismutase, and S-nitrosothiols (SNOs), which have recently been identified in brain. However, the chemical biology of NO- remains largely unknown. Here, we show that NO- -unlike NO*, but reminiscent of NO+ transfer (or S-nitrosylation)- -reacts mainly with Cys-399 in the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor to curtail excessive Ca2+ influx and thus provide neuroprotection from excitotoxic insults. This effect of NO- closely resembles that of NOS, which also downregulates NMDA receptor activity under similar conditions in culture.

Lad, an adapter protein interacting with the SH2 domain of p56lck, is required for T cell activation.

T cell-specific Src family tyrosine kinase, p56lck, plays crucial roles in T cell differentiation, activation, and proliferation. These multiple functions of p56lck are believed to be conducted through the protein-protein interactions with various cellular signaling proteins. To clarify the mechanisms through which p56lck contributes to T cell signaling, we identified the proteins binding to the Src homology 2 (SH2) domain of p56lck through a tyrosine phosphorylation-dependent yeast two-hybrid screening. Subsequent characterization of positive clones revealed the presence of a protein of 366 aa named Lad (Lck-associated adapter protein), which is a potential murine homologue of previously reported TSAd, a T cell-specific adapter protein. Lad contains several protein-protein interaction domains including a zinc-finger motif, an SH2 domain, a proline-rich SH3 binding motif, and several phosphotyrosine sites. Furthermore, Lad was tyrosine phosphorylated and associated with p56lck in vivo and redistributed from cytoplasm to the plasma membrane in a T cell activation-dependent manner. Moreover in T cells, IL-2 promoter activity was enhanced upon coexpression of Lad but was inhibited by the coexpression of antisense Lad RNA. These characteristics of Lad suggest that Lad play an essential role as an adapter protein in p56lck-mediated T cell signaling.

Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor.

Zinc (Zn2+) inhibition of N-methyl-D-aspartate receptor (NMDAR) activity involves both voltage-independent and voltage-dependent components. Recombinant NR1/NR2A and NR1/NR2B receptors exhibit similar voltage-dependent block, but voltage-independent Zn2+ inhibition occurs with much higher affinity for NR1/NR2A than NR1/NR2B receptors (nanomolar versus micromolar IC50, respectively). Here, we show that two neighboring histidine residues on NR2A represent the critical determinant (termed the "short spacer") for high-affinity, voltage-independent Zn2+ inhibition using the Xenopus oocyte expression system and site-directed mutagenesis. Mutation of either one of these two histidine residues (H42 and H44) in the extracellular N-terminal domain of NR2A shifted the IC50 for high-affinity Zn2+ inhibition approximately 200-fold without affecting the EC50 of the coagonists NMDA and glycine. We suggest that the mechanism of high-affinity Zn2+ inhibition on the NMDAR involves enhancement of proton inhibition.

Cysteine carboxyl O-methylation of human placental 23 kDa protein.

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.

Neuroprotective versus neurodestructive effects of NO-related species.

Nitric oxide (NO.) can lead to damaging or protective actions in the central nervous system. Here we consider the chemistry of the NO group and its redox-related species that can lead to these exactly opposite ends. In the neurodestructive mode, NO. reacts with superoxide anion (02.-) to form peroxynitrite (ONOO-), which leads to neuronal injury. In contrast, the reaction of the NO group with cysteine sulfhydryls on the NMDA receptor leads to a decrease in receptor/channel activity. avoidance of excessive Ca2+ entry, and thus neuroprotection. Site-directed mutagenesis of recombinant NMDA receptor subunits has recently increased our knowledge of such redox modulation by NO. Transfer of the NO group to cysteine sulfhydryls on the NMDA receptor or other proteins, known as S-nitrosylation, is becoming recognized as a ubiquitous regulatory reaction, skin to phosphorylation, and represents a form of redox modulation in diverse tissues including the brain.

Redox modulation of the NMDA receptor by NO-related species.

The chemical reactions of NO are largely dictated by its redox state. Increasing evidence suggests that the various redox states of the NO group exist endogenously in biological tissues. In the case of NO+ equivalents, the mechanism of reaction often involves S-nitrosylation (transfer of the NO group to a cysteine sulfhydryl to form an RS-NO); further oxidation of critical thiols can possibly form disulfide bonds. We have physiological and chemical evidence that NMDA receptor activity can be modulated by S-nitrosylation, resulting in a decrease in channel opening. Recent data suggest that NO-, probably in the singlet (or high-energy) state, can also react with critical sulfhydryl group(s) of the NMDA receptor to down-regulate its activity; in the triplet (lower-energy) state NO- may oxidize these NMDA receptor sulfhydryl groups by formation of an intermediate such as peroxynitrite. It has also been reported that NO can react with thiol but only under specific circumstances, e.g., if an electron acceptor such as O2 is present, as well at catalytic amounts of metals like copper, and if the conditions do not favor the kinetically preferred reaction with O2.- to yield peroxynitrite. Mounting evidence in many fields suggests that S-nitrosylation can regulate the biological activity of a great variety of proteins, perhaps analogous to phosphorylation. Thus, this chemical reaction is gaining acceptance as a newly-recognized molecular switch to control protein function via reactive thiol groups such as those encountered on the NMDA receptor.

Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor.

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine's neurotoxic (agonist) attributes at 10-100 microM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.