Effects of acivicin on growth, mycotoxin production and virulence of phytopathogenic fungi.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 µmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.

Raloxifene inhibits menin-dependent estrogen receptor activation in breast cancer cells.

BACKGROUND: Menin is a tumor suppressor encoded by Men1 that is mutated in the human-inherited tumor syndrome--multiple endocrine neoplasia type 1. Menin binds to estrogen receptors (ER) to enhance estrogen activity in breast cancer cells. AIM: Our clinical study showed that the outcome in the case of menin-positive tumors was worse than in the case of menin-negative tumors. We examined the role of raloxifene on the cell growth in a menin-positive breast cancer cell line. MATERIAL AND METHODS: To examine the mechanism of raloxifene on menin-dependent activation of ER, we employed the mammalian two-hybrid system. We have established a breast cancer cell line that stably expresses menin. Using these cells, we have examined the effect of raloxifene and tamoxifen on cell growth of menin-transfected cells. RESULTS: The expression of activation function (AF)-2 enhanced menin-mediated luciferase expression in the mammalian two-hybrid assay. Raloxifene attenuated the effect of menin on estrogen response element-luciferase activation, indicating that raloxifene inhibited the binding of menin to AF-2. Raloxifene significantly inhibited the growth of menin-transfected cells in a dose-dependent manner. Tamoxifen also inhibited menin-transfected MCF-7 cells; however, this inhibition was much less than that of raloxifene. CONCLUSION: Raloxifene inhibits the binding of menin to the AF-2 domain of ERα, suggesting that raloxifene is one of the therapeutic options for menin-positive breast cancer.

Hyperglycemia suppresses ABCA1 expression in vascular smooth muscle cells.

Hyperglycemia is a major risk factor for atherosclerotic disease. The ATP-binding cassette transporter A1 (ABCA1) functions as a pivotal regulator of lipid efflux from cells to apolipoproteins and is thus involved in lowering the risk of atherosclerosis. In this study, we have examined the glucose-mediated regulation of the ABCA1 gene expression in vascular smooth muscle cells. ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and reporter gene assay. The results showed that the expression of the ABCA1 mRNA and protein decreased after the cells were treated with 22.4 mM glucose for 48 h. The transcriptional activity of the ABCA1 promoter paralleled the endogenous expression of the ABCA1 gene. Next, we used inhibitors of certain signal transduction pathways to demonstrate that the glucose-induced ABCA1 suppression is sensitive to the p38-mitogen-activated protein kinase (MAPK) inhibitors. The expression of a constitutively active form of p38-MAPK in the cells inhibited the ABCA1 promoter activity, irrespective of the presence of glucose. A dominant-negative mutant of p38-MAPK abrogated the inhibitory effect of glucose on the ABCA1 promoter activity. These results indicate that the glucose-induced suppression of ABCA1 expression is partially mediated by the activation of the p38-MAPK pathway.

Interferon alpha decreases expression of human scavenger receptor class BI, a possible HCV receptor in hepatocytes.

BACKGROUND: Infection with the hepatitis C virus (HCV) causes acute hepatitis. This disease has a high probability of becoming chronic and leading to cirrhosis, but a more deadly consequence is hepatocellular carcinoma. Interferon alpha (IFN alpha)-based treatment combined with ribavirin is the major therapeutic choice available for the treatment of chronic HCV infection. AIMS: The scavenger receptor class B type I (SR-BI) or its human homologue CD36 and LIMPII Analogous-1 (hSR-BI/CLA-1) has recently been shown to interact with HCV envelope glycoprotein E2, thus suggesting that it might participate in entry of the virus into host cells. This rationale underlies current interest in the potential role of IFN alpha in hSR-BI/CLA-1 expression in HepG2 cells. RESULTS: It was shown that endogenous hepatocyte expression of hSR-BI/CLA-1 was suppressed by exposure to IFN alpha. Decreased hSR-BI/CLA-1 expression in IFN alpha-treated cells was due to lower transcriptional activity of the promoter. A potential pathway for the effect of IFN alpha on hSR-BI/CLA-1 promoter activity was identified when the inhibitory action of IFN was abrogated in signal transducer and activator of transcription 1 (STAT1)/STAT2 knocked-down cells. Exposure of HepG2 cells to IFN alpha elicited a rapid phosphorylation of STAT1/STAT2, a known target of IFN alpha signalling. In addition, the mutagenesis of a STAT1/STAT2 response element in the hSR-BI/CLA-1 promoter abolished the ability of IFN alpha to suppress promoter activity. CONCLUSIONS: Together, these results indicate that the STAT1/STAT2 pathway participates in IFN alpha inhibition of hSR-BI/CLA-1 expression, and raise the possibility that lowering the expression of this gene may be of therapeutic value for treating HCV infections.

The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves.

The FAD7 gene, a gene for a chloroplast [omega]-3 fatty acid desaturase, is responsible for the trienoic fatty acid (TA) formation in leaf tissues. The TA content of the leaf tissue of the 25[deg]C-grown transgenic tobacco (Nicotiana tabacum cv SR1) plants, in which the FAD7 gene from Arabidopsis thaliana was overexpressed, increased uniformly by about 10%. Fatty acid unsaturation in all major leaf polar lipid species increased in the 25[deg]C-grown FAD7 transformants but was approximately the same between the control plants and the FAD7 transformants when grown at 15[deg]C. Therefore, the overexpression of the exogenous FAD7 gene leads to the same consequence in the tobacco plants as the low-temperature-induced TA production that may be catalyzed by an endogenous, temperature-regulated chloroplast [omega]-3 fatty acid desaturase. In the 25[deg]C-grown control plants, the chilling treatment caused symptoms of leaf chlorosis and suppression of leaf growth. The 25[deg]C-grown FAD7 transgenic plants conferred alleviation of these chilling-induced symptoms. A reductions of the chilling injury similar to that of the FAD7 transformants was also observed in the 15[deg]C-preincubated control plants. These results indicate that the increased TA production during chilling acclimation is one of the prerequisites for the normal leaf development at low, nonfreezing temperatures.

Secretion and clinical significance of atrial natriuretic peptide in patients with muscular dystrophy.

The plasma concentration of atrial natriuretic peptide was measured in patients with muscular dystrophies to study its relationship with congestive heart failure. In patients with Duchenne muscular dystrophy, the plasma atrial natriuretic peptide concentration was 35.5 +/- 3.3 pg/mL (mean +/- SE), which was higher than that in age-matched normal subjects (9.8 +/- 0.6 pg/mL). It increased with progression of disability and showed significant correlations with the cardiothoracic ratio and the ratio of the preejection period to the left ventricular ejection time. In patients with other types of muscular dystrophy, the plasma atrial natriuretic peptide concentration showed no significant change. Immunohistochemical examination demonstrated many atrial natriuretic peptide-positive cells in atrial muscle of an autopsied patient, indicating preservation of the peptide until the end stage. These findings suggest that measurement of the plasma atrial natriuretic peptide concentration is useful for evaluating heart failure in patients with Duchenne muscular dystrophy.

Plasma atrial natriuretic peptide in patients with congenital heart diseases.

The plasma level of human alpha-atrial natriuretic peptide was measured in healthy children and patients, 1 month to 15 years of age, with congenital heart diseases. Significant increases were found in patients with a ventricular septal defect, tricuspid valve atresia, patent ductus arteriosus, and atrial septal defect but not in those with pulmonary valve stenosis or tetralogy of Fallot. The levels were significantly higher in children with ventricular septal defects (221 +/- 123 pg/mL) or patent ductus arteriosus (124 +/- 38 pg/mL) than in those with atrial septal defects (65 +/- 42 pg/mL) (P less than .01). The increased levels appeared to be correlated with enlargement of the left atrium (r = .85, P less than .01) but not with the right atrial size or the mean right atrial pressure. They were higher in younger than in older healthy infants, but this age difference did not affect the results. These findings indicate that human alpha-atrial natriuretic peptide is released into the circulation in response to chronic atrial expansion in patients with congenital heart disease and may have an important role in volume homeostasis.

Radioimmunoassay for atrial natriuretic peptide: method and results in normal subjects and patients with various diseases.

A sensitive and specific radioimmunoassay for alpha-human atrial natriuretic polypeptide (alpha-hANP) was developed to determine its plasma level. Anti-alpha-hANP rabbit serum was specific for the N-terminus and ring structure of alpha-hANP, and showed no appreciable cross-reactions with other neuropeptides. The lowest level of alpha-hANP detectable by this radioimmunoassay was 4 pg per tube. The intra- and inter-assay coefficients of variation were 4.6-11.4% and 7.9-11.8%, respectively, and the recovery rates at 4 concentrations were 62.6-74.0%. The fasting plasma alpha-hANP concentration in normal subjects were 19.3 +/- 1.0 ng/l (mean +/- SE; n = 54), and there was no sex difference. The plasma alpha-hANP level in normal subjects fell significantly during water deprivation and increased significantly on infusion of hypertonic saline. The mean plasma levels of alpha-hANP were higher than normal in patients with essential hypertension, liver cirrhosis, congestive heart failure and chronic renal failure. Our results indicate that this radioimmunoassay is suitable for determining the alpha-hANP concentration in human plasma and can assess changes in pathological and physiological states.

Enhanced secretion and impaired natriuretic action of atrial natriuretic peptide in response to hypertonic saline infusion in patients with essential hypertension.

To clarify the natriuretic action of endogenous atrial natriuretic peptide (ANP) in patients with essential hypertension (EHT), we examined the relationship between ANP release and urinary sodium excretion in response to hypertonic saline infusion. Plasma ANP levels increased from 13.3 +/- 2.0 pg/ml to 37.0 +/- 3.0 pg/ml in patients with EHT and from 9.2 +/- 1.5 pg/ml to 21.1 +/- 4.0 pg/ml in normal subjects after the infusion. The area under the curve (AUC) of plasma ANP response was significantly higher in patients with EHT than in normal subjects (P < 0.05). There was a significant positive correlation between AUC and urinary sodium excretion in both groups (P < 0.01). However, the ratio of urinary sodium excretion to AUC was significantly lower in patients with EHT than in normal subjects (P < 0.01). These results suggest that impaired natriuretic response to endogenous ANP is one of the factors responsible for the development of EHT and that enhanced secretion of ANP in response to hypertonic saline infusion is a compensatory mechanism to the impaired natriuretic action of ANP in patients with EHT.

Different secretion profiles of atrial and brain natriuretic peptides after acute volume loading in patients with ischemic heart disease.

In order to clarify the different secretion profiles of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in response to acute hemodynamic change by volume expansion, we measured plasma ANP and BNP levels after intravenous isotonic saline infusion for 3 min at a rate of 3 ml/kg body weight/min in 15 patients with ischemic heart disease. Plasma ANP and BNP levels before the volume loading were 30.7 +/- 16.7 and 19.4 +/- 24.6 pg/ml, respectively. Five and 10 minutes after infusion, plasma ANP levels rose significantly to 43.5 +/- 20.7 and to 46.0 +/- 22.5 pg/ml, respectively (p < 0.01), and plasma BNP levels rose significantly to 27.3 +/- 30.8 and 24.8 +/- 23.2 pg/ ml, respectively (p < 0.01). The BNP/ANP ratio was not affected by volume loading. The maximum increments of plasma ANP level correlated significantly with those of the mean pulmonary capillary wedge pressure (mPCWP, r = 0.78, p < 0.01) or left ventricular end-diastolic pressure (LVEDP, r = 0.86, p < 0.01). However, there were no significant correlations between the maximum increments of plasma BNP levels and those of mPCWP or LVEDP. Plasma ANP level can be a useful parameter for atrial pressure even if the hemodynamic state change rapidly. However, in an early phase of ventricular overload BNP secretion is not increased sufficiently despite the raised LVEDP, and plasma BNP level may not always reflect ventricular hemodynamics.

Histopathological studies of a case of cytomegalovirus retinochoroiditis.

A case of cytomegalovirus (CMV) retinochoroiditis initially misdiagnosed as fungal endophthalmitis is reported. An 83-year-old man who was suspected of having cholangiocarcinoma presented uveitis in both eyes. Candida endophthalmitis was suspected on the basis of ophthalmic findings and past history, which included systemic corticosteroid administration and intravenous hyperalimentation. Intravenous treatment with miconazole was not effective. At autopsy, 3 months after the initial ophthalmological examination, the right eye was enucleated and examined histologically and histochemically. Light microscopic examination showed extensive retinal necrosis and numerous cytomegalic cells, so-called owl's eye cells, with intranuclear and intracytoplasmic inclusion bodies. CMV particles were seen by electron microscopy, and CMV-infected cells were observed by immunohistochemical staining by the direct method with fluorescein-labeled antibodies. These findings indicate that in suspected cases of fungal endophthalmitis various tests should also be carried out for CMV.

[Secretion and clinical significance of atrial natriuretic peptide in patients with muscular dystrophy].

In patients with Duchenne muscular dystrophy (DMD), heart failure appears in later stage of the disease due to myocardial degeneration and respiratory insufficiency, and sometimes causes death. However, there have been no adequate parameters which can be used easily to evaluate the grade of heart failure in DMD, except cardiac enlargement and pulmonary congestion observed by chest X-ray picture. Thus, we measured the plasma concentrations of atrial natriuretic peptide (ANP) in the patients with muscular dystrophy of various types, and studied a relationship between plasma ANP concentration and heart failure, expecting that it could be an index of heart failure in DMD patients. The plasma ANP concentrations in patients with DMD were 35.5 +/- 3.3pg/ml (mean +/- SE) and higher than in normal subjects (19.3 +/- 1.0pg/ml). In the patients with limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy and neurogenic muscular atrophy, the plasma ANP concentration showed a tendency to elevate. However, no elevation of plasma ANP levels was observed in the patients with other types of muscular dystrophy. In DMD, number of the patients having a high plasma ANP concentration was increased with progress of disability grade, and decrease in serum creatine kinase activity and serum myoglobin concentration. There was a significant correlation (p less than 0.01) between plasma ANP concentration and cardiothoracic ratio or PEP/LVET, but no correlation between the concentration and respiratory failure. Immunohistochemistry of the atrial cardiac muscle of an autopsied DMD case revealed many ANP-positive atrial muscle cells, indicating the preservation of ANP-secreting function.(ABSTRACT TRUNCATED AT 250 WORDS)

[Comparison of buprenorphine and fentanyl for postoperative pain relief by continuous epidural infusion].

This study examined analgesic efficacy and adverse effects of buprenorphine and fentanyl for the postoperative pain relief by continuous epidural infusion. Fifty patients after upper or lower abdominal surgeries were assigned to two groups and buprenorphine and fentanyl were epidurally administered postoperatively. Buprenorphine (B) group received bolus injection of B 0.1mg + saline 8 ml and continuous infusion of B 0.8 mg+saline 92 ml (2 ml.h-1). Fentanyl group received bolus injection of F 0.1 mg+saline 6 ml and continuous infusion of F 0.6 mg+saline 84 ml (2 ml.h-1). There was no significant difference between the two groups in the analgesic efficacy, which became lower from 2 to 12 hours postoperatively. However, compared with buprenorphine group, the incidence of nausea or vomiting and dizziness was significantly less in the fentanyl group (11 vs. 4 cases and 7 vs. 1 cases). These results imply that the major site of action of epidurally administered fentanyl is the spinal cord. In contrast, analgesic effect of epidural buprenorphine appears to be enhanced by the supraspinal action. We conclude that fentanyl is superior to buprenorphine for postoperative pain relief by continuous epidural infusion.

[Postoperative pain relief by continuous epidural infusion: a comparison of three solutions].

The effect of three solutions on postoperative pain relief by continuous epidural infusion was studied. Seventy-five patients after upper or lower abdominal surgeries were assigned to one of three groups and the postoperative pain relief was evaluated for 48 hours. Group I: bolus injection of buprenorphine (Bn) 0.1 mg + saline (S) 8 ml and continuous infusion (2 ml.h-1) of Bn 0.8 mg + S 92 ml; Group II: bolus injection of Bn 0.1 mg + 0.5% bupivacaine (Bc) 4 ml + S 4 ml and continuous infusion of Bn 0.8 mg + S 92 ml; Group III: bolus injection of Bn 0.1 mg + 0.5% Bc 4 ml + S 4 ml and continuous infusion of Bn 0.8 mg + 0.5% Bc 40 ml + S 60 ml. The combination of buprenorphine with low-dose bupivacaine (Group III) offered the most effective postoperative analgesia in three groups without increasing the frequency of adverse effects induced by epidural administration of local anesthetics. However, even in Group III, the percentage of patients complaining of pain at bed rest was still high during early postoperative period (56% at 6 hours postoperatively). The results suggest that further consideration is necessary on agents selection and dosage adjustment for the postoperative epidural analgesia.

[Preoperative diagnosis of unroofed coronary sinus: a case report].

A 54-year-old man was admitted to our hospital because of exertional dyspnea. A second heart sound with fixed splitting and a systolic ejection murmur along the left sternal border was audible. The chest roentgenogram showed increased pulmonary vascularity, and the electrocardiogram showed incomplete right bundle branch block. Two-dimensional echocardiography in the parasternal view demonstrated a partition defect between the left atrium and the coronary sinus. Furthermore, transesophageal echocardiography revealed a left-to-right shunt flow into the coronary sinus through the defect. At these points, the patient was diagnosed as having a partially unroofed mid-portion of the coronary sinus. Unroofed coronary sinus is a cardiac anomaly rarely diagnosed prior to surgical operation. Two-dimensional echocardiography, especially transesophageal echocardiography, is useful for the preoperative diagnosis of unroofed coronary sinus.

Effects of hypergravity stimulus on global gene expression during reproductive growth in Arabidopsis.

The life cycle of higher plants consists of successive vegetative and reproductive growth phases. Understanding effects of altered gravity conditions on the reproductive growth is essential, not only to elucidate how higher plants evolved under gravitational condition on Earth but also to approach toward realization of agriculture in space. In the present study, a comprehensive analysis of global gene expression of floral buds under hypergravity was carried out to understand effects of altered gravity on reproductive growth at molecular level. Arabidopsis plants grown for 20-26 days were exposed to hypergravity of 300 g for 24 h. Total RNA was extracted from flower buds and microarray (44 K) analysis performed. As a result, hypergravity up-regulated expression of a gene related to ß-1,3-glucanase involved in pectin modification, and down-regulated ß-galactosidase and amino acid transport, which supports a previous study reporting inhibition of pollen development and germination under hypergravity. With regard to genes related to seed storage accumulation, hypergravity up-regulated expression of genes of aspartate aminotransferase, and down-regulated those related to cell wall invertase and sugar transporter, supporting a previous study reporting promotion of protein body development and inhibition of starch accumulation under hypergravity, respectively. In addition, hypergravity up-regulated expression of G6PDH and GPGDH, which supports a previous study reporting promotion of lipid deposition under hypergravity. In addition, analysis of the metabolic pathway revealed that hypergravity substantially changed expression of genes involved in the biosynthesis of phytohormones such as abscisic acid and auxin.

A marine phytoplankton (Prymnesium parvum) up-regulates ABC transporters and several other proteins to acclimatize with Fe-limitation.

Iron (Fe) is one of the vital limiting factors for phytoplankton in vast regions of the contemporary oceans, notably the high nutrient low chlorophyll regions. Therefore, it is apparent to be acquainted with the Fe uptake strategy of marine phytoplankton under Fe-limited condition. In the present study, marine phytoplankton Prymnesium parvum was grown under Fe-deplete (0.0025 µM) and Fe-rich (0.05 µM) conditions, and proteomic responses of the organism to Fe conditions were compared. In sodium dodecyl sulfate (SDS) gel electrophoresis, 7 proteins (16, 18, 32, 34, 75, 82, and 116 kDa) were highly expressed under Fe-deplete condition, while one protein (23 kDa) was highly expressed under Fe-rich condition. These proteins were subjected to 2-dimensional gel electrophoresis (2-D DIGE) to differentiate individual proteins, and were identified by matrix-assisted laser desorption-ionization-time of flight-mass spectrometer (MALDI-TOF-MS) analysis. The results showed that under Fe-deplete condition P. parvum increases the biosynthesis of ATP binding cassette (ABC) transporters, flagellar associated protein (FAP), and Phosphoribosylaminoimidazole-succinocarboxamide synthase. These proteins are assumed to be involved in a number of cellular biochemical processes that facilitate Fe acquisition in phytoplankton. Under Fe-deplete condition, P. parvum increases the synthesis of ribulose biphosphate carboxylase (RuBisCo), malate dehydrogenase, and two Fe-independent oxidative stress response proteins, manganese superoxide dismutase (MnSOD) and Serine threonine kinase (STK). Thus, marine phytoplankton may change their Fe acquisition strategy by altering the biosynthesis of several proteins in order to cope with Fe-limitation.

Quantitative laser atom probe analyses of hydrogenation-disproportionated Nd-Fe-B powders.

We report a successful atom probe tomography of hydrides in hydrogenation-disproportionated Nd-Fe-B powder using a green femtosecond laser. The atom probe specimens were prepared from one particle of powder using the focused ion beam lift-out method. The atom probe tomography taken from an α-Fe/NdH(2) structure suggested that B and Ga (trace added element) were partitioned in the NdH(2) phase. The hydrogen concentration of 64 at% determined from the atom probe analysis was in excellent agreement with the stoichiometry of the NdH(2) phase.