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Endoscopic hematoma evacuation is an established treatment option for patients with intracerebral hemorrhage (ICH). Although it has the advantages of being less invasive than craniotomy and more effective than conservative therapy, its role and indication in ICH treatment are still controversial. We retrospectively reviewed 34 cases with ICH treated by endoscopic surgery. ICH patients with mild-to-moderate consciousness disturbance were treated by endoscopic surgery. Of 34 patients, 24 (70.6%) exhibited significant improvement in their consciousness level immediately after surgery. Of 29 patients other than those with cerebellar hemorrhage, 12 (41.4%) showed significant functional recovery in their motor weakness on discharge. The extent of improvement in consciousness level was positively correlated to that in motor weakness. The removal rate of hematoma was 68.4±31.7% on average and was not correlated to the extent of functional recovery. Two patients experienced re-bleeding after operation and had poor functional prognoses. Of these, 1 was administered anticoagulants and the other was given antiplatelets before operation. These data suggest that endoscopic hematoma evacuation may be beneficial for ICH patients with mild-to-moderate consciousness disturbance by promptly improving their consciousness level, leading to the final functional recovery. On the other hand, endoscopic hematoma evacuation has the potential problem of low hemostatic ability. The operative indication for patients taking an anticoagulant or antiplatelet drugs should be

We have previously shown that the AEX-1 protein, which is expressed in postsynaptic muscles, retrogradely regulates presynaptic neural activity at the Caenorhabditis elegans neuromuscular junctions. AEX-1 is similar to vertebrate Munc13-4 protein, suggesting a function for vesicle exocytosis from a kind of cells. Compared to emerging evidences of the role of Munc13 proteins in synaptic vesicle release, however, the precise mechanism for vesicle exocytosis by AEX-1 and Munc13-4 is little understood. Here we have identified SYN-1 as a candidate molecule of AEX-1-dependent vesicle exocytosis from non-neuronal cells. The syn-1 gene encodes a C. elegans syntaxin, which is distantly related to the neuronal syntaxin UNC-64. The syn-1 gene is predominantly expressed in non-neuronal tissues and genetically interacts with aex-1 for presynaptic activity. However, the two proteins did not interact physically in our yeast two-hybrid system and mutational SYN-1 did not bypass the requirement of AEX-1 for the behavioral defects in aex-1 mutants, whereas mutant UNC-64 does in unc-13 mutants. These results suggest that a novel molecular interaction between the AEX-1 and syntaxin may regulate vesicle exocytosis for retrograde signal release.

Na + /K + ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential. Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy. However, its precise function at the synapse is unknown. Here we show that two mutations in the α subunit of the eat-6 Na + /K + ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine (Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ). These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na + /K + ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a pump activity-independent, allele-specific role for Na + /K + ATPase on postsynaptic organization and synaptic efficacy.

Periodic behavioral motor patterns are normally controlled by neural circuits, such as central pattern generators. We here report a novel mechanism of motor pattern generation by non-neural cells. The defecation motor program in Caenorhabditis elegans consists of three stereotyped motor steps with precise timing and this behavior has been studied as a model system of a ultradian biological clock [J.H. Thomas, Genetic analysis of defecation in C. elegans , Genetics 124 (1990) 855–872; D.W. Liu, J.H. Thomas, Regulation of a periodic motor program in C. elegans , J. Neurosci. 14 (1994) 1953–1962; K. Iwasaki, D.W. Liu, J.H. Thomas, Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans , Proc. Natl. Acad. Sci. USA 92 (1995), 10317–10321]. It was previously implied that the inositol-1,4,5-trisphosphate (IP3) receptor in the intestine was necessary for this periodic behavior [P. Dal Santo, M.A. Logan, A.D. Chisholm, E.M. Jorgensen, The inositol trisphosphate receptor regulates a 50 s behavioral rhythm in C. elegans , Cell 98 (1999) 757–767]. Therefore, we developed a new assay system to study a relationship between this behavioral timing and intestinal Ca 2+ dynamics. Using this assay system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca 2+ -wave propagation in the intestine. Lack of the Ca 2+ -wave propagation correlated with no coordination of the motor steps in the CaMKII mutant. Also, when the Ca 2+ -wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated, which phenocopied ablation of the motor neurons AVL and DVB. These observations suggest that an intestinal Ca 2+ -wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans .

The alkaline condition is an important factor for the alkaline single-cell gel electrophoresis (SCG) assay to detect the genotoxic effects of chemicals. In order to understand the relationship between DNA migration and alkaline condition, the effect of 13 model chemical mutagens with different modes of action was evaluated with the alkaline SCG assay under two different alkaline conditions (pH 12.1 and 12.6). CHO cells were sampled just after treatment for 1 h. The X-ray mimetic mutagen BLM increased DNA migration at pH 12.1 and 12.6 and the results were the same at both pH values. Six alkylating mutagens MNU, ENU, MNNG, ENNG, MMS, and EMS and one base adduct inducer 4-NQO induced a dose-dependent response only at pH 12.6. Two DNA crosslinking agents, MMC and DDP, and AMD had negative results. MMC and DDP, however, reduced the positive response of BLM, suggesting that DNA crosslinks could be detected. These results demonstrated that the alkaline condition was important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals.

C. elegans aex-3mutations cause pleiotropic behavioral defects that are suggestive of reduced synaptic transmission. aex-3mutations also show strong genetic interactions with mutations in unc-31and unc-64, two other genes implicated in synaptic transmission. Physiological and pharmacological studies indicate that aex-3defects are presynaptic. In aex-3mutants, the synaptic vesicle–associated RAB-3 protein aberrantly accumulates in neuronal cell bodies and is reduced in synapse-rich axons. This localization defect is specific to RAB-3, since other synaptic proteins are localized normally in aex-3mutants. aex-3encodes a 1409 amino acid protein with strong homology to DENN, a human protein of unknown function. In C. elegans, aex-3is expressed in all or nearly all neurons. These results suggest that AEX-3 is a novel regulator of presynaptic activity that interacts with RAB-3 to regulate synaptic vesicle release.

Aims To study the time and costs of insulin treatment of newly registered outpatients with Type 2 diabetes mellitus (T2DM). Methods In total, 355 patients with T2DM were registered on their first visit to one of 11 diabetes clinics across Japan. Of these, 313 were not being treated with insulin (the non-insulin group), whereas 42 were (the insulin group). In the insulin group, 26 were already on insulin at the first visit, whereas 16 were started on insulin after their first visit. The time and costs involved in the care were recorded over the following 5 months. Results In the first 3 months, considerable time was expended in both groups, with the time spent by physicians a little (but significantly) longer for the insulin group. The total time expended by all care providers was approximately 1.3-fold greater for the insulin compared with the non-insulin group. The total cost and total cost/min for the insulin group was almost twice that for the non-insulin group. Over the 5-month period, mean HbA1c in the non-insulin group improved from 8.0% to 6.5%, with 72% achieving a glycemic target of HbA1c ≤ 6.5%. In contrast, in the insulin group, mean HbA1c improved from 9.4% to 7.6%, with only 39% achieving the target. There were no reports of major hypoglycemic events in either group and body mass index remained stable. Conclusions The insulin therapy for T2DM can be achieved safely and effectively at outpatient clinics, even though it requires considerably more time and resources than non-insulin therapy.

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1.

✓ Morphological and ultrastructural changes in normal mouse brain tissue were investigated after intracerebral stereotactic injections of tumor necrosis factor (specific activity: 2.0 × 106U/mg protein) into the right frontal lobe. The mice received either a single infusion or multiple tumor necrosis factor infusions in three different dose groups (10, 100, or 500 U). Compared with sham-treated control mice that received adjusted intracerebral injections of purified albumin, the tumor necrosis factor-treated mice in all dose groups did not show any specificin vivobehavioral abnormalities during the 2 months of study following the infusions.Histological studies revealed hemorrhage attributable to the mechanics of the intracerebral infusions, a thickening of the arachnoid membranes, a reactive gliosis, and neutrophilic and/or mononuclear cell infiltration along the infusion pathway. A local neutrophilic response was prominent 1 day after tumor necrosis factor injection. An immunohistochemical analysis indicated that the mononuclear cell infiltration consisted of lymphocytes and macrophages. Except for the transient neutrophilic infiltration, these histological alterations did not differ from those seen in the sham-treated control groups, and most nonspecific reactive changes disappeared within 8 weeks after the injections. Furthermore, an ultrastructural study showed no apparent pathological changes in the cytoplasmic organelles of neuronal, glial, and endothelial cells in the tumor necrosis factor-injected mouse specimens. These results suggest that the tumor necrosis factor injections caused no specific toxicity and did not alter the parenchymal and stromal cells comprising normal mouse brain tissue.

The clonal analysis in the ultrastructure of tumor-lymphocyte interaction was carried out in order to investigate the precise mechanism responsible for CTL-mediated cytolysis of tumor cells. A glioma-derived cell line (GI-1) and autologous tumor-specific cytotoxic T lymphocyte (CTL) clones were established. The CTL lines were composed of the morphologically homogeneous lymphocytes with intracytoplasmic electron-dense secretory granules. After the stimulation by GI-1, the size of the CTLs increased, and the intracytoplasmic organellas were developed. It was noted that the intracytoplasmic secretory granules markedly increased in number and size, and many of them exhibited an “immature” appearance. On the other hand, the tumor cells underwent a progressive degeneration. In contrast, the stimulation by other antigens caused only small morphological changes in the CTLs. It is suggested, therefore, that the secretory function of tumor-specific CTLs is activated by the stimulation of the specific antigen, and that soluble factors in the secretory granules in the CTLs may be closely associated with the mechanism of target Cell lysis.

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μmax) and the maximum specific production rate of ethanol (qmax) were 0.12 h−1 and 0.1 g ethanol/109 cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 109 cells/ml for production of 85 g/l ethanol at a dilution rate (D1) of 0.2 h−1. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D1) of 0.2 h−1 by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 109 cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (Dt) of 0.25 h−1, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (Dt) of 0.25 h−1 was achieved, just as it was in the laboratory-scale fermentation test.

Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesicle-mediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor–green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

SummaryThe transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg2+ levels, while GTL-1 mainly contributes to appropriate Mg2+ responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg2+ levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.

The extent to which excitable cells and behavior modulate animal development has not been examined in detail. Here, we demonstrate the existence of a novel pathway for promoting vulval fates in C. elegansthat involves activation of the heterotrimeric Gαq protein, EGL-30. EGL-30 acts with muscle-expressed EGL-19 L-type voltage-gated calcium channels to promote vulva development, and acts downstream or parallel to LET-60 (RAS). This pathway is not essential for vulval induction on standard Petri plates,but can be stimulated by expression of activated EGL-30 in neurons, or by an EGL-30-dependent change in behavior that occurs in a liquid environment. Our results indicate that excitable cells and animal behavior can provide modulatory inputs into the effects of growth factor signaling on cell fates,and suggest that communication between these cell populations is important for normal development to occur under certain environmental conditions.

Guanine nucleotide exchange is essential for Rab GTPase activities in regulating intracellular vesicle trafficking. This exchange process is facilitated by guanine nucleotide exchange factor (GEF). Previously, we identified Caenorhabditis elegans AEX-3 as a GEF for Rab3 GTPase. Here we demonstrate that AEX-3 regulates neural activities through a second, previously unrecognized pathway via interactions with the novel protein CAB-1. CAB-1 is 425 amino acids long and has an 80 amino acid motif in common with the mouse neural protein NPDC-1. cab-1 and rab-3 mutants have different behavioral defects, and RAB-3 localization and function are apparently normal in cab-1 mutants, indicating that the CAB-1 pathway is distinct from the RAB-3 pathway. The aex-3 mutant phenotype resembles the sum of the rab-3 and cab-1 mutant phenotypes, indicating that AEX-3 regulates two different pathways for neural activities. We propose that connection of multiple pathways may be an important feature of Rab GEFs to coordinate various cellular events.

✓ Eleven lymphocyte clones were established from the peripheral blood lymphocytes of a patient with gliosarcoma by means of autologous tumor stimulation and the limiting-dilution technique with recombinant interleukin-2. Ten of the 11 clones were cytotoxic against the autologous tumor cell line GI-1. Seven of the 10 clones were also cytotoxic against allogeneic brain-tumor lines and HeLa cells, one clone was cytotoxic against several target cells, and two clones were specifically cytotoxic against GI-1 and allogeneic brain-tumor cells. One of the 11 clones was not cytotoxic against any target cells tested. Lymphokine-activated killer cells induced by recombinant interleukin-2 alone exhibited cytotoxic activity against all target tumor cells tested. Surface phenotypic analysis revealed that all lymphocyte clones expressed CD3 antigen, some expressed CD4 antigen, and others expressed CD8 antigen. These clones seemed to be antigen-specific cytotoxic T lymphocyte clones. Analysis with these antigen-specific cytotoxic T lymphocyte clones may be useful in the elucidation of tumor-specific or tumor-associated antigens on autologous tumor cells.

Retrograde signaling from postsynaptic cells to presynaptic neurons is essential for regulation of synaptic development, maintenance, and plasticity. Here we report that the novel protein AEX-1 controls retrograde signaling at neuromuscular junctions in C. elegans . aex-1 mutants show neural defects including reduced presynaptic activity and abnormal localization of the synaptic vesicle fusion protein UNC-13. Muscle-specific AEX-1 expression rescues these defects but neuron-specific expression does not. AEX-1 has an UNC-13 homologous domain and appears to regulate exocytosis in muscles. This retrograde signaling requires prohormone-convertase function in muscles, suggesting that a peptide is the retrograde signal. This signal regulates synaptic vesicle release via the EGL-30 Gqα protein at presynaptic terminals.

Summary Intercellular calcium waves can be observed in adult tissues, but whether they are instructive, permissive, or even required for behavior is predominantly unknown. In the nematode Caenorhabditis elegans , a periodic calcium spike in a pacemaker cell initiates a calcium wave in the intestine [1, 2]. The calcium wave is followed by three muscle contractions that comprise the defecation motor program [1]. Normal wave propagation requires the pannexin gap-junction subunit INX-16 at the interfaces of the intestinal cells. In the absence of this gap-junction subunit, calcium waves are frequently absent. The remaining waves are slow, initiate at abnormal locations, or travel in the opposite direction. Abnormal waves are associated with parallel effects in the first step of the motor program: The contractions of the overlying muscles fail to propagate beyond the pacemaker cell, are slow, initiate in abnormal locations, or are reversed. Moreover, the last two motor steps are predominantly absent. Finally, the absence of this gap-junction subunit also affects the reliability of the pacemaker cell; cycle timing is often irregular. These data demonstrate that pannexin gap junctions propagate calcium waves in the C. elegans intestine. The calcium waves instruct the motor steps and regulate the pacemaker cell's authority and reliability.

✓ The authors have investigated whether specific pathological changes and antibodies against interleukin-2 (IL-2) are induced after intracerebral administration of recombinant IL-2 (rIL-2). In addition, IL-2 receptor (IL-2R) expression was checked on the cell surface of normal brain tissues before and after the intracerebral infusion. Reconstituted rIL-2 (specific activity 1.2 × 107 U/mg protein) was injected into the right cerebral hemisphere of normal adult C57BL/6 mice in three different dose groups, each receiving single or multiple infusions of 8, 32, or 80 U. In sham control experiments, mouse albumin purified by gel filtration and ion exchange chromatography and adjusted to the same concentration of protein as rIL-2 was injected into mice at various doses. Anti-IL-2 antibodies were measured by an enzyme-linked immunosorbent assay concurrently with assessment of IL-2 activity in serum. The IL-2R expression was determined by using immunofluorescence techniques with monoclonal antibodies against mouse IL-2R. Since histological alteration after rIL-2 injection did not differ from that in the sham control preparations, it seems that there is no direct toxic action of rIL-2 on normal brain tissues. Interleukin-2 antibodies were produced at low levels only in mice injected repeatedly at the maximum dose, and levels were insignificant in other groups. Serum levels of IL-2 activity remained low. The IL-2R expression within the brain was not enhanced within 8 weeks following the intracerebral administration of rIL-2, suggesting that direct intracerebral infusion of rIL-2 may be safely used in the immunotherapy of brain tumors.