A Virulence-Reducing Mutation in the Postharvest Citrus Pathogen Alternaria citri.

ABSTRACT Alternaria citri causes Alternaria black rot, a postharvest fruit disease, on a broad range of citrus cultivars. We previously described that an endopolygalacturonase minus mutant of A. citri caused significantly less black rot in citrus fruit. To search for other essential factors causing symptoms in addition to endopolygalacturonase, a random mutation analysis of pathogenicity was performed using restriction enzyme-mediated integration. Three isolates among 1,694 transformants of A. citri had a loss in pathogenicity in a citrus peel assay, and one of these three mutants was a histidine auxotroph. Gene AcIGPD that encodes imidazole glycerol phosphate dehydratase, the sixth enzyme in the histidine biosynthetic pathway, was cloned, and the mutant containing the disrupted target gene, AcIGPD, caused less black rot.

Green Fluorescent Detection of Fungal Colonization and Endopolygalacturonase Gene Expression in the Interaction of Alternaria citri with Citrus.

ABSTRACT Alternaria citri, a postharvest pathogen, produces endopolygalacturonase (endoPG) and causes black rot on citrus fruit. We previously described that an endoPG-disrupted mutant of Alternaria citri was significantly reduced in its ability to macerate plant tissue and cause black rot symptoms on citrus. In order to investigate colonization of citrus fruit tissues by Alternaria citri, pTEFEGFP carrying a green fluorescent protein (GFP) gene was introduced into wild-type Alternaria citri and its endoPG-disrupted mutant (M60). Green fluorescence was observed in spores, germ tubes, appressoria, and infection hyphae of transformants G1 (derived from wild type) and GM4 (derived from M60). Hyphae of G1 but not GM4 vertically penetrated the peel, but the hyphae of both G1 and GM4 spread equally in the juice sac area of citrus fruit. Green fluorescence of Alternaria citri transformant EPG7 carrying a GFP gene under control of the endoPG gene promoter of Alternaria citri was induced by pectin in the peel during the infection stage, but repressed completely in the juice sac area, likely by carbon catabolite repression by sugars in the juice.

Involvement of the brain-derived neurotrophic factor/TrkB pathway in neuroprotecive effect of cyclosporin A in forebrain ischemia.

Recent studies have shown that cyclosporin A, a specific antagonist of calcineurin, a phosphatase, ameliorates neuronal cell death in the CA1 sector of the hippocampus after forebrain ischemia in animal models. The mechanism of this neuroprotective effect, however, has not yet been established. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, is one of the potent survival and developmental factors whose expression is regulated by cyclic AMP-response element-binding protein (CREB). Activation of CREB is dependent on its phosphorylation at Ser(133), and calcineurin has been reported to dephosphorylate CREB via protein phosphatase 1. Based on these observations, we attempted to investigate how cyclosporin A treatment would affect the changes of phosphorylated CREB (pCREB), BDNF and its receptor tyrosine kinase B (TrkB) after forebrain ischemia in rats. Phosphorylation of CREB was kept augmented throughout the time course examined in cyclosporin A-treated animals, while it ceased without cyclosporin A. Reverse transcription-polymerase chain reaction revealed prolonged maintenance of BDNF mRNA expression in the CA1 sector of cyclosporin A-treated animals. The protein expression of BDNF and TrkB appeared to be up-regulated in cyclosporin A-treated animals, whereas it was transiently up-regulated but decreased to the marginal level of expression without cyclosporin A.From these results we suggest that cyclosporin A induces pCREB by an inhibition of calcineurin, resulting in the induction of BDNF. The mechanisms by which cyclosporin A protects the CA1 region from neuronal cell death in forebrain ischemia may involve the interaction of pCREB, BDNF and TrkB.

[Whole body heat production as a quantitative marker of anesthetic management under general anesthesia].

In order to evaluate the effects of the rapid induction and the slow induction of general anesthesia on the management of anesthesia and the circadian rhythm, the time-dependent whole body heat production in mice (ICR, male, 6 to 10 weeks after birth) was measured by calorimeter. In the single administration of either an i.v. anesthetic (thiamylal) or a volatile anesthetic (isoflurane), the minimum heat production and its duration time were similar. In the co-administration of i.v. and volatile anesthetics, the minimum heat production was significantly lower (P < 0.01) and the duration of this minimum production was significantly more sustained (P < 0.01) compared to those seen in the single administration of each anesthetic. Moreover, by measurement of the whole body heat production, the slow induction of general anesthesia markedly affected the circadian rhythm on the next postoperative day. These results indicate that the measurement of the whole body heat production will be a marker for the recovery from general anesthesia and the return to the usual life style (such as the QOL).

Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata.

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.

Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis.

Fibronectin performs essential roles in embryonic development and is prominently expressed during tissue repair. Two forms of fibronectin have been identified: plasma fibronectin (pFn), which is expressed by hepatocytes and secreted in soluble form into plasma; and cellular fibronectin (cFn), an insoluble form expressed locally by fibroblasts and other cell types and deposited and assembled into the extracellular matrix. To investigate the role of pFn in vivo, we generated pFn-deficient adult mice using Cre-loxP conditional gene-knockout technology. Here we show that pFn-deficient mice show increased neuronal apoptosis and larger infarction areas following transient focal cerebral ischemia. However, pFn is dispensable for skin-wound healing and hemostasis.

[Comparative effects of autologous blood transfusion and isovolemic colloids transfusion on hemorrhagic shock: measurement of NO production and tissue oxygen pressure].

Effects of autologous blood transfusion and isovolemic colloids transfusion on hemorrhagic shock model in rats were investigated. The hemorrhagic shock model in rats was prepared by rapid exsanguination and the blood pressure was kept at 40 mmHg for 30 min. As a marker of hemodynamic change, the blood gas analysis was performed, and the tissue oxygen pressure of the liver and the spleen was also measured. Furthermore, we analyzed the changes in NO production in the liver and the spleen of the hemorrhagic shock model. By hemorrhagic shock, Ht, HCO3-, base excess, and PaO2 levels decreased, and liver NO level, but not spleen NO, increased rapidly. However, the liver PtO2 did not show any changes during hemorrhagic shock, although the spleen PtO2 tended to decrease. These changes seen at the hemorrhagic shock were equally restored after the treatment with either autologous blood transfusion or isovolemic colloids transfusion. These results indicate that either autologous blood transfusion or isovolemic colloids transfusion can be useful for the therapy of severe hemorrhagic shock, and the NO production in the liver can participate in the maintenance of homeostasis under hemorrhagic shock.

[Changes in brain metabolites under combined mild-hypothermia and isoflurane anesthesia in rat with temporal brain ischemia].

The significance of mild-hypothermia as a treatment of brain ischemia-induced neuronal cell death was investigated by measurement of hippocampul excitatory amino acids, brain lactate and energy-charge just after 10 min of transient forebrain ischemia in a rat model with four vessels occluded. After 10 min of ischemia, cerebral circulation was restored. At that time, in the control group transient increases of hippocampul aspartate and glutamic acid levels were observed. Furthermore, brain lactate levels were also elevated but the energy-charge was reduced. These significant changes were observed in the non-isoflurane anesthesia and mild-hypothermia rats (control group). However, in rats with treatment of either isoflurane or mild-hypothermia, the excessive amount of amino acids and the significant fluctuation of brain metabolic/energy pathway seen in the control group were suppressed. Particularly, in the combined treatment group, these increased and decreased phenomena induced by brain ischemia were significantly inhibited. In a group of pre- and post-treatment of mild-hypothermia, the maximum peak of lactate was significantly less than that seen in the control group, although the sustained increased level of lactate was detected. These results indicate that the combined treatment with isoflurane anesthesia and mild-hypothermia is a suitable treatment for the brain dysfunction induced by ischemia and that the sustained hypothermia may help restore brain lactate levels after brain ischemia because of the lasting anaerobic metabolism.

Abdominal wall lift versus carbon dioxide insufflation for laparoscopic resection of ovarian tumors.

STUDY OBJECTIVE: To evaluate and compare changes in pulmonary mechanics and stress hormone responses between abdominal wall lift (gasless) and carbon dioxide (CO2) insufflation laparoscopic surgery during controlled general anesthesia. DESIGN: Prospective, randomized clinical study. SETTING: Operating rooms at a university medical center. PATIENTS: 12 ASA physical status I and II female patients undergoing laparoscopic resection of ovarian tumors. INTERVENTIONS: Patients were divided into two groups of six each: the abdominal wall lift group and the CO2 pneumoperitoneum laparoscopic group. Following induction of anesthesia, patients were paralyzed and the trachea was intubated. Anesthesia was maintained with isoflurane and nitrous oxide (N2O) in oxygen. Throughout the procedure, patients were mechanically ventilated with a tidal volume of 10 ml/kg and a respiratory rate of 10 breaths per minute. MEASUREMENTS AND MAIN RESULTS: During the laparoscopic procedure, arterial blood gases, acid-base balance, pulmonary mechanics, stress-related hormones, and urine output were measured and recorded. In the CO2 pneumoperitoneum group, arterial CO2 tension increased (p < 0.01), dynamic pulmonary compliance decreased (p < 0.01), peak inspiratory airway pressure increased (p < 0.01), and plasma epinephrine (p < 0.05), norepinephrine (p < 0.05), dopamine (p < 0.01), and antidiuretic hormones (p < 0.05) increased significantly during the laparoscopic procedure as compared to the abdominal lift group. Adrenocorticotropic hormone and cortisol increased as compared to baseline value in both groups (p < 0.05). Urine output was significantly less (p < 0.01) in the CO2 pneumoperitoneum group than in the abdominal wall lift group. CONCLUSIONS: Abdominal wall lift laparoscopic surgery is physiologically superior to CO2 pneumoperitoneum laparoscopic surgery as seen during the conditions of this study. Abdominal wall lift laparoscopic surgery provides normal acid-base balance and a lesser degree of hormonal stress responses, it maintains urine output, and it avoids derangement of pulmonary mechanics.

Increased production of nitric oxide metabolites in the hippocampus under isoflurane anaesthesia in rats.

There is disagreement concerning the role of nitric oxide (NO) in general anaesthesia. The present study was conducted to determine whether the anaesthetic drug isoflurane alters levels of NO metabolites, NOx (NO2 and NO3), in the hippocampus of rats during and after anaesthesia. Results showed resting hippocampal NOx levels of about 20 pmol in freely moving control rats. Five minutes after the induction of isoflurane anaesthesia (4.72% = 4 minimum alveolar concentrations, detected by righting reflex), there was loss of the righting reflex coincident with a significant elevation in hippocampal NOx levels. During isoflurane anaesthesia, the maximum NOx concentration rose approximately 2.4-fold higher than control levels; the NO3 level increased about 5-fold higher than resting levels. NOx levels returned to control levels following discontinuation of the anaesthetic. When rats were pretreated with L-NG-nitro arginine methyl ester, an NO synthase-inhibitor, the isoflurane-induced increases in NOx were markedly suppressed. D-NG-nitro arginine methyl ester was ineffective in preventing these neurochemical changes, thus indicating the stereo-selective nature of the inhibition by L-NG-nitro arginine methyl ester Furthermore, L-NG-nitro arginine methyl ester, pre-treatment likewise prevented increases in both NO2 and NO3 levels. When rats were exposed to 80% nitrous oxide in oxygen, there was loss of the righting reflex but no change in hippocampal NOx levels. These findings indicate that isoflurane increases production of hippocampal NO and that this may be pertinent to general anaesthetic drug effects.

Neuroprotective properties of propofol and midazolam, but not pentobarbital, on neuronal damage induced by forebrain ischemia, based on the GABAA receptors.

BACKGROUND: The mechanism of the neuroprotective effects of propofol was compared to two other types of intravenous (i.v.) anesthetics (i.e., benzodiazepine; midazolam and barbiturate; pentobarbital) using Mongolian gerbils focusing on GABA receptor subtypes. METHODS: Neuronal injury was induced by a 4-min occlusion of the common carotid arteries followed by reperfusion. One week after occlusion, animals were transcardially perfused for histochemistry. Neuronal death in four brain regions was evaluated by direct visual counting of acidophilic neurons. RESULTS: Seven days after this ischemic episode, severe neuronal injury was measured in the hippocampal CA1 area (> 98% of total cells damaged) and parietal cortex (> 35%). Also lateral thalamus and caudate putamen were damaged but to a lesser extent (about 10%). The neuronal injury in these areas was significantly attenuated by propofol, midazolam and the GABAA agonist, muscimol, intraperitoneally administered 15 min prior to ischemia. This neuroprotective property, however, was lacking with pentobarbital and GABAB agonist baclofen. Concomitant pretreatment with subthreshold doses of propofol and muscimol significantly reduced the amount of cell death induced by brain ischemia. On the other hand, pretreatment with the GABAA antagonist bicuculline significantly inhibited the neuroprotective effects of propofol. However, a GABAB antagonist, phaclofen, was without effect on neuronal damage and on neuronal protection of propofol. CONCLUSION: These results indicate that activation of GABAA receptors, which include the specific binding subunits for propofol and midazolam, but not pentobarbital, plays a role in the inhibition of neuronal death induced by brain ischemia.

[Clinical study of AJ-007 (bupivacaine) in spinal anesthesia--investigation of clinical dosage of isobaric and hyperbaric formulations].

Spinal anesthesia with two types of 0.5% bupivacaine hydrochloride solution, isobaric AJ-007 I and hyperbalic AJ-007 H, was studied clinically in 106 surgical patients with collaboration of 7 university hospitals. The following results were obtained. 1) Successful analgesia with motor block on abdominal wall and legs could be induced by dosages of 2.0 ml, 3.0 ml and 4.0 ml of either of the two solutions. 2) With isobaric solutions, the duration of the block was prolonged dose dependently. Anesthetic levels tended to increase and onset times of the block tended to decrease, with increasing dosage. 3) Hyperbalic solutions tended to produce higher levels of the block more rapidly. However, the duration of the block seemed to be shorter than that achieved by isobaric solutions. 4) The incidence of hypotension and bradycardia was similar to that observed in ordinary clinical spinal anesthesia. In one young male, a high level (C 2) of anesthesia was achieved after administration of 4.0 ml of hyperbaric solution. This patient was managed uneventfully under general anesthesia during the surgery. These results indicate that these two solutions of 0.5% bupivacaine are useful for spinal anesthesia.

[Effects of midazolam and propofol on dopamine release from rat striatal slice using a fast-cyclic voltammetry system].

In order to evaluate the difference in pharmacological mechanism between midazolam and propofol, we focused on the interaction between dopaminergic and GABAergic neurons in the striatum because of its important role in the regulation of motor system and arousal response, and examined these inhibitory effects on dopamine (DA) release induced by high-K from the rat striatal slice using the fast-cyclic voltammetry method. Between 0.1 and 200 nmol, the standard curve of DA was linear. The peak and the sensitivity of DA oxidation were different from those of norepinephrine and DA main metabolites. The dosages between 0.1 and 10 micro M of propofol significantly blocked the high-K evoked DA release, although the dosage of larger than 50 micro M of propofol potentiated DA release. In case of midazolam, the dosages between 0.1 and 50 micro M markedly suppressed DA release induced by high-K in a dose-dependent manner. The recovery time of DA release after removal of midazolam from incubation medium was longer than that seen in the treatment of propofol. In conclusion, propofol and midazolam inhibited high-K evoked DA release from striatal slice, although these efficacies were dissimilar. Furthermore the pharmacological effects of larger dosage of propofol was different from those obtained by its smaller dosages. These results suggest that the anesthetic actions of propofol and midazolam are partially related to inhibition of DA neuron A1 activity and that the excitatory symptom induced by a larger dose of propofol may be related to its potantiation of DA release.

[The influence of ulinastatin on renal function during anesthesia for renal transplantation].

The purpose of this study was to evaluate the influence of ulinastatin (UST) on renal function and changes in granulocyte elastase during anesthesia in 10 living related and 10 cadaveric renal transplantations. UST (300,000 units) was administered after the induction of anesthesia and renal vessel anastomosis, respectively. The living patients who received UST had greater urine volume during the operative and postoperative periods. In addition, the granulocyte elastase values of these patients showed relative suppressive changes during the intraoperative and postoperative periods. In cadaveric renal transplant patients, UST did not influence serum BUN or creatinine, although urine volume was greater in the UST-treated patients than in the patients who did not receive. We could not conclude that the intraoperative administration of UST affected the renal function of renal transplants, but we did find that UST is useful to suppress granulocyte elastase which is concerned with the index of stress or inflammation. It is necessary to investigate further the method of administration of UST for renal transplantation.

[Three case reports of the use of stellate ganglion block for the climacteric psychosis].

There are many reports of the use of stellate ganglion block (SGB) for the climacteric psychosis, which is considered to be sympathicotonic response to stress. We experienced three cases of the SGB therapy for the climacteric psychosis. We performed SGB three times per week by 1% lidocaine 5 ml, and observed improvements of the symptoms after doing SGB for five times. The patients reported psychological relaxation after receiving SGB therapy. We examined the changes of the serum concentrations of ACTH, LH, FSH, and catecholamines (epinephrine, norepinephrine) before and after SGB in 8 patients who were suffering from climacteric psychosis, because we wanted to know the endocrinological response to SGB. We observed a significant decrease in norepinephrine concentration after SGB, which is reasonable considering the sympathetic blockage. There were no significant changes of ACTH, LH, FSH, and epinephrine. We conclude that SGB therapy must be effective for the climacteric psychosis because of sympathetic blockade. But we could not clarify the influence of endocrinological response to SGB.

[Hemodynamic changes secondary to overload infusion during cadaveric renal transplantation--comparison between nitrous oxide-isoflurane anesthesia and continuous epidural anesthesia].

The hemodynamic status of renal transplant patients is important for achieving early diuresis. Many reports have demonstrated that overload infusion can reduce the frequency of acute tubular necrosis (ATN). We studied the effect of overload fluid infusion using pulmonary arterial pressure (PAP) and pulmonary wedge pressure (PCWP) monitoring on forty patients undergoing cadaveric renal transplantation. Patients were divided into two groups. Group 1 received general anesthesia by nitrous oxide-isoflurane. Group 2 received continuous epidural anesthesia. Mean PAP > 15 mmHg and mean PCWP > 10 mmHg were achieved with the infusions of normosmotic saline, colloid solution and human albumin. Systolic arterial pressure (SAP) > 150 mmHg was achieved by intravenous administration of dopamine when required. Hemodynamic stability and diuresis in the early postoperative period were maintained in both groups. Group 1 required lower doses of dopamine than Group 2. Blood loss and infusion requirements were lower in group 1 than in group 2. No patient in either group developed pulmonary edema. We conclude that overload infusion using PAP and PCWP monitoring during general anesthesia can achieve safe diuresis immediately after cadaveric renal transplantation.

[Cardiovascular effects of, and catecholamine response to, high dose fentanyl or NLA in patients for valve replacement].

We measured the cardiovascular effect of, and catecholamine and other hormonal responses to, anesthetic doses of fentanyl and original NLA in 25 patients for open heart surgery. The patients were randomly divided into three groups (group N, F30, F75). During induction, in group N; droperidol 0.25 mg.kg-1 and fentanyl 5 micrograms.kg-1, in group F30; fentanyl 30 micrograms.kg-1, and in group F75; fentanyl 75 micrograms.kg-1 were administered intravenously. Additional fentanyl was administered at a rate of 100 to 200 micrograms.h-1. Droperidol 0.25 mg.kg-1 was administered in group N when cardiopulmonary bypass (CPB) was disconnected. Plasma samples were assayed for norepinephrine, epinephrine, ACTH and cortisol before and after induction, during sternotomy, 60 minutes after institution of CPB, after weaning from CPB, and before as well as after extubation. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and rate pressure product (RPP) were calculated simultaneously at the blood samplings. In all groups, no remarkable change in cardiovascular dynamics was observed. CPB was associated with marked increases in catecholamines, but high dose fentanyl in dose of 75 micrograms.kg-1 was able to suppress epinephrine level more than in group N. In high dose fentanyl group (F30, F75) ACTH was within normal ranges, even during CPB. The results suggest that high dose fentanyl is a complete anesthetic in patients for cardiac surgery. But a large dose of fentanyl causes small decreases in heart rate and arterial blood pressure. Our data indicate that group F30 is an attractive anesthetic technique for patients with valvular disease.

[Changes in granulocyte elastase caused by the use of ulinastatin during anesthesia for renal transplantation].

In patients with renal failure, releases of chemical mediators having inhibitory influences on organs and circulatory system have been observed. Therefore, in long-term dialysis patients, it is necessary to consider the biocompatibilities of the equipment for dialysis. On 13 cases of renal transplantations, we measured the pre- and post-operative changes of granulocyte elastase (GE) and evaluated the effect of ulinastatin. The renal transplanted patients receiving a long-term dialysis showed higher GE values than that of normal patients even before the operation. During the anesthesia, GE increased gradually and further increased after the operation. In patients given ulinastatin during anesthesia, the increase in GE during the operation was suppressed but an increase after the operation was observed. The renal transplanted patients are easily infectible. Therefore, it is useful to measure GE pre- and post-operatively for earlier diagnosis of the postoperative infection and septicemia. The administration of ulinastatin can inhibit the increase of GE. It seems that ulinastatin increases renal blood flow and improves renal function.

[A comparison of induced hypotensive anesthesia using trimetaphan, prostaglandin E1 and nicardipine for neurosurgery].

We performed hypotensive anesthesia using trimetaphan, prostaglandin E1, or nicardipine for eighteen patients undergoing cerebral aneurysm clipping. We measured the hemodynamic parameters (systolic arterial pressure, diastolic arterial pressure, and heart rates), concentration of catecholamines in blood (epinephrine, norepinephrine), blood platelet counts, and aggregation of blood platelets, before induction of anesthesia, before induced hypotension, during hypotension, after hypotension, and at the end of operation. Hypotension was produced rapidly, and hemodynamics was stable with any of the three drugs. In trimetaphan group, however, recovery time of arterial pressure was longer than in other two groups. There was no significant change of catecholamine during induced hypotension in prostaglandin E1 and nicardipine groups. However, in trimetaphan group, concentration of norepinephrine in blood decreased significantly during hypotension. No significant change was observed in either platelet count or platelet aggregation. These results suggest that trimetaphan has an effect to suppress excretion of stress hormones, and prostaglandin E1 and nicardipine have no influence on blood catecholamine levels and aggregation of platelet. We consider that nicardipine is useful as a hypotensive drug for neurosurgery.