Preliminary development and evaluation of the support system for care of mechanically ventilated patients.

BACKGROUND: We wanted to demonstrate the feasibility of a novel computer-assisted ventilator alarm system, the support system for care of mechanically ventilated patients (SCMVP), to detect gas leaks and provide graphical information on the site of the leak in a manikin model. METHODS: We tested six leakage scenarios. Four scenarios were applied to both the respiratory circuits with the SCMVP and without the SCMVP (conventional system), and two scenarios were each specific to one of the systems. Fifteen registered nurses were asked to manage three scenarios each (two mutual and one system-specific scenario). Time to identify the site of the leak was measured and compared between the two systems. RESULTS: The SCMVP showed significantly shorter time for troubleshooting in one of the four mutual scenarios and shorter accumulated time for troubleshooting in the four mutual scenarios [18.0 (range, 14.5-19.5) and 48.5 (9.0-180.0) s, respectively] compared with the conventional system [76.0 (47.0-133.8) and 82.5 (16.0-180.0) s, respectively]. In the mutual scenarios, SCMVP resulted in significantly more frequent incidences of successful troubleshooting within 30 s and less frequent incidences of troubleshooting requiring >180 s [43.3% (13/30) and 6.7% (2/30), respectively] compared with the conventional system [13.3% (4/30) and 30% (9/30), respectively]. CONCLUSIONS: The SCMVP can facilitate rapid and successful recognition of the site of leak in a respiratory circuit in a simulation environment.

Hemoglobin Hijiyama: a new fast-moving hemoglobin in a Japanese family.

A variant of hemoglobin A, named Hb Hijiyama, found in two generations of a Japanese family living in Hiroshima, Japan, has a higher anodal electrophoretic mobility than hemoglobin A; a gain of two negative charges per molecule is indicated. Fingerprinting and amino acid analysis showed the biochemical anomaly to be in the beta chain at residue 120, where lysine is replaced by glutamic acid. In the heterozygote carriers of the abnormal hemoglobin there is no apparent association with clinical or hematologic abnormalities.

Hemoglobin Hiroshima (beta-143 histidine--aspartic acid): a newly identified fast moving beta chain variant associated with increased oxygen affinity and compensatory erythremia.

During a survey for hemoglobinopathies in over 9000 residents of Hiroshima Prefecture, Japan, a fast moving hemoglobin was identified in eight members of three generations in a Japanese family. The abnormal hemoglobin, named Hb Hiroshima, constitutes about 50% of the total hemoglobin in hemolysates from the carriers who have a mild erythremia but are otherwise apparently clinically unaffected. All preparations of Hb Hiroshima have increased affinity for oxygen, by either tonometric or oxygen electrode determinations. At pH 7.0, the oxygen pressure, P(50) required to half saturate an unfractionated hemolysate from a carrier was one-half that of Hb A, and the P(50) of a purified sample containing no Hb A was one-fourth that of Hb A. The pH dependence of the oxygen equilibrium (Bohr effect) is below normal, as shown by the absolute value of the Bohr effect factor which is about half that of Hb A, in the pH range between 7.0 and 7.4. The Hill constant, n, for Hb Hiroshima between pH 7.0 and 7.4 is 2-2.4, compared to 2.8-3 for Hb A under the same conditions, indicating reduction of, but not complete abolition of heme-heme interaction. Urea dissociation and canine hybridization tests located the biochemical lesion in the beta chain. Fingerprints (Ingram), carboxypeptidase digestion, and amino acid analysis demonstrated that the substitution was at residue 143 in the beta chain, where histidine was replaced by aspartic acid.In contrast to other recently described high oxygen affinity mutants that show intact Bohr effects, all three of the major characteristics of the reversible combination of hemoglobin with oxygen (oxygen equilibrium, heme-heme interaction, and pH dependence) are affected in Hb Hiroshima. A tentative interpretation of these effects, relating structure to function, is offered in terms of recently developed models of normal hemoglobin.

Immediate elimination of labile HbA1c with allosteric effectors of hemoglobin.

Our aim was to find a simple method of removing labile glycosylated hemoglobin (HbA1c) from blood samples before it is measured by cation-exchange chromatography. Labile HbA1c is formed by the binding of glucose to the NH2-terminal valine of the beta-chain of HbA. We sought a more competitive binder for the same site to dissociate labile HbA1c to glucose and HbA. Inorganic phosphates were found to have a strong allosteric effect and a great ability to eliminate labile HbA1c. We developed our method with 4 mM tetrapolyphosphate in the hemolyzing solution to eliminate labile HbA1c during the automatic processing (at pH 6 and heated for 2 min at 45 degrees C) of blood samples for HbA1c estimation. This may be useful when estimating HbA1c by the manual method.

Crystallization and X-ray studies of the DNA-binding domain of OmpR protein, a positive regulator involved in activation of osmoregulatory genes in Escherichia coli.

The OmpR protein of Escherichia coli is a positive regulator involved in the activation of expression of ompC and ompF genes encoding the major outer membrane protein OmpC and OmpF, respectively. The C-terminal half domain of OmpR (OmpR-C), which is responsible for DNA-binding, has been crystallized using the hanging drop vapour diffusion method. X-ray studies show that the crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with a = b = 60.4 A, c = 58.8 A and gamma = 120 degrees. The asymmetric unit contains one molecule. The crystals diffract to at least 3 A resolution and are suitable for X-ray structure analysis.

Randomized study of orally administered fluorinated pyrimidines (capecitabine versus S-1) in women with metastatic or recurrent breast cancer: Japan Breast Cancer Research Network 05 Trial.

PURPOSE: Capecitabine and S-1 are orally administered fluorinated pyrimidines with high-level activity against metastatic breast cancer (MBC). This randomized, multicenter, phase II study compared the activities and safeties of the oral fluoropyrimidines, capecitabine and S-1, in breast cancer patients. METHODS: Patients with MBC were randomly assigned to receive capecitabine 825 g/m(2) twice daily on days 1-21 every 4 weeks or S-1 40-60 mg twice daily, according to body surface area, on days 1-28 every 6 weeks. The primary endpoint was progression-free survival (PFS). RESULTS: A total of 142 patients were enrolled and randomized to either capecitabine (N = 73) or S-1 (N = 69). Median PFS (progression-free survival) was 1.2 years for capecitabine and 1.3 years for S-1, with a hazard ratio (S-1/capecitabine) of 0.85 (95 % confidence interval [CI] 0.52-1.38) (P = 0.48 by log-rank). The confirmed objective response rates were 24.0 % for capecitabine and 23.1 % for S-1 (P = 0.938). The most common treatment-related adverse events were grade 1-2 in intensity. Thrombocytopenia (S-1: 9.2 %, capecitabine: 1.4 %; P = 0.040) and nausea (S-1: 26.2 %, capecitabine: 14.1 %; P = 0.079) were more frequent in the S-1 group, while hand-foot syndrome occurred more often in the capecitabine group (S-1: 10.8 %, capecitabine: 25.4 %; P = 0.029). CONCLUSIONS: The results of the current study demonstrate that both S-1 and capecitabine are effective and well-tolerated treatments in patients with MBC, while their adverse events were different. They are both convenient, orally administered drugs, making them attractive agents for use in outpatient treatment.

Alpha-Fetoprotein and hepatitis B antigen in hepatocarcinogenesis.

Blood from 394 unselected autopsy cases was examined for HB Ag, HB Ab and AFP. Liver morphology of 71 cases of cirrhosis with hepatoma and 32 cases of cirrhosis without hepatoma was studied in detail and correlated to HB Ag, HB Ab, and AFP. Significantly lowered humoral immunity to HB Ag exposure was established for the cirrhosis with hepatoma cases. The exposure rate for both cirrhosis cases with hepatoma and cirrhosis cases without hepatoma was the same (48%), but about 80% of each exposed group were either HB Ag or HB Ab positive. The cirrhosis with hepatoma group tended to be HB Ag positive and the cirrhosis without hepatoma group tended to be HB Ab positive. The lowered immune response seems to be specific to the hepatoma association, because the group with neoplasms other than the hepatoma reacted exactly the same as the group of the cirrhosis without hepatoma. Twenty-five per cent of the cirrhosis with hepatoma were associated with inactive cirrhosis and 75% were associated with active cirrhosis. Seventy-two per cent of the inactive cirrhosis cases with hepatoma were exposed to HB Ag, but only 42% of the active cirrhosis cases with hepatoma were exposed to HB Ag. On the morphological basis, the inactive cirrhosis was interpreted as an impaired cellular immunity and the active cirrhosis as a delayed hypersensitivity reaction. The possibility was discussed that both are important factors in the development of hepatoma preceded by cirrhosis. AFP tends to be positive in the inactive cirrhosis with hepatoma as well as HB Ag, but the relationship between AFP and HB Ag for hepatocarcinogenesis needs further investigation.

Nucleotide sequence, cloning, and overexpression of the D-threonine dehydrogenase gene from Pseudomonas cruciviae.

D-Threonine dehydrogenase (EC 1.1.1) catalyses the oxidation of the 3-hydroxyl group of D-threonine. The nucleotide sequence of the structural gene, dtdS, for this enzyme from Pseudomonas cruciviae IFO 12047 was determined. The dtdS gene encodes a 292 amino acid polypeptide. The enzyme was overproduced in Escherichia coli cells; the activity was found in cell extracts of the clone. The enzyme showed high sequence similarity to 3-hydroxyisobutyrate dehydrogenases. This is the first example showing the primary structure of an enzyme catalysing the NADP(+)-dependent dehydrogenation of D-threo-3-hydroxyamino acids.

Further cases of Hb Hirosaki in two Japanese families.

Hb Hirosaki (alpha 43(CE1)Phe----Leu) was recently found anew in two Japanese families. The probands had chronic hemolytic disorders, while two other carriers of the same Hb in one family did not exhibit increased hemolysis. Hb Hirosaki comprised 5 and 2% of the total Hb in the splenectomized and non-splenectomized proband, respectively, while it comprised only 1% in the two clinically silent carriers. The expected nucleotide substitution was found in the alpha 2 (rather than alpha 1) globin gene in 1 proband. The abnormal globin was synthesized (with incorporation of [3H]-leucine) at about 30 and 20% of the total alpha-chain in reticulocytes of the probands and in those of the silent carriers, respectively. The specific radioactivity of the abnormal chain was about 20-times higher than that of the normal counterpart in the non-splenectomized patient and silent carriers (7-times in the splenectomized patient). The total alpha/beta radioactivity ratio was much higher than unity. The newly synthesized Hb Hirosaki had a slightly high isoelectric point and appeared just in front of normal Hb A in anion-exchange chromatography, but the electric charge of aged Hb Hirosaki was indistinguishable from that of Hb A. These results suggest that Hb Hirosaki is quite unstable, and is rapidly denatured and degraded after biosynthesis. The physical number of the alpha globin genes was normal in all subjects.

Angiotensin II type 1 receptor-mediated increase in cytosolic Ca(2+) and proliferation in mesothelial cells.

We investigated the Ca(2+) signaling pathways of the response to angiotensin II in pleural mesothelial cells and the role of these Ca(2+) signaling pathways in mesothelial cell proliferation. Rat pleural mesothelial cells were maintained in vitro, and the Ca(2+) movement to angiotensin II was evaluated using the fluorescent Ca(2+) indicator fura 2. Furthermore, proliferation of mesothelial cells was assessed using a spectrophotometric 3-(4, 5-dimethylthazol-2-yl)-2,5-diphenyl-2H-tetrasodium bromide (MTT) assay. Angiotensin II (1 pM-100 microM) induced in mesothelial cells a biphasic elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) that consisted of a transient initial component, followed by a sustained component. Neither removal of extracellular Ca(2+) nor inhibition of Ca(2+) influx by 1 microM nifedipine affected the angiotensin II-induced initial transient elevation of [Ca(2+)](i) in mesothelial cells. Nifedipine did not block angiotensin II-induced sustained elevation of [Ca(2+)](i). Angiotensin II (1 pM-100 microM) had a proliferative effect on mesothelial cells in a dose-dependent manner. Angiotensin II type 1 (AT(1)) receptor antagonist ([Sar(1), Ile(8)]angiotensin II) inhibited both angiotensin II-induced elevation of [Ca(2+)](i) and proliferation of mesothelial cells. Pertussis toxin did not affect angiotensin II-induced responses. These results suggest that angiotensin II-induced responses to mesothelial cells are extremely dependent on the angiotensin AT(1) receptor coupled with pertussis toxin-insensitive G protein.

Involvement of BMP-2 signaling in a cartilage cap in osteochondroma.

This study describes the distributions of bone morphogenetic protein (BMP)-2 as well as mRNAs for BMP receptor type IB (BMPRIB). collagen types II (Col II) and III (Col III) in a growing "cartilage cap" of osteochondroma. In situ hybridization and immunohistochemical study were performed using histological sections obtained during surgery. BMP-2 was detected in mesenchymal cells in the outer fibrous layer and chondrocytes in the inner cartilaginous matrix, positive for Col III and Col II, respectively. BMPRIB mRNA was distributed in chondrocytes. This is the first study to provide observational evidence of the involvement of BMP-2 signaling in the pathogenesis of cartilage cap of osteochondroma. and suggests the role of BMP-2 in the growth of cartilage cap in osteochondroma.

Source of elevated serum mitochondrial creatine kinase activity in patients with malignancy.

Mitochondrial creatine kinase (CK-mit) is increased in cancer tissues of the digestive tract. There is no difference in molecular weight, electrophoretic and kinetic properties between the isoenzymes extracted from the tumor and those from the adjacent normal tissues. High non-CK-M/total CK activity ratios in some sera from cancer patients probably reflect leakage of CK-mit from the tumor tissues.

Frequency of alkaline phosphatase-immunoglobulin complex among diseased and healthy populations.

Serum alkaline phosphatase-immunoglobulin complex was surveyed by polyacrylamide gel electrophoresis. Its frequency among apparently healthy blood donors was 1/3,015 or 0.03%. A significantly higher frequency, 8/2,400 or 0.33%, was observed among patients under 60 years of age. There was an apparently age related, sharp increase among patients over 60 years.

Urea polyacrylamide gel electrophoresis of PCMB precipitate as a sensitive test for the detection of the unstable hemoglobin subunit.

A sensitive test for the detection of the abnormal subunit of unstable hemoglobins is described. The unstable hemoglobin subunit was selectively precipitated by paramercuribenzoic acid treatment of the carboxy hemolysate, and the precipitate was studied by urea polyacrylamide gel electrophoresis for globin composition. The test enabled the detection of an electrophoretically silent, alpha-chain unstable variant comprising only 1% of total hemoglobin. Other applications included the demonstration of Hb Köln in a cord blood, and the detection and purification of very slightly unstable hemoglobin variants.

Effects of naloxone on cardiac energy metabolism in acute hemorrhage in rats.

It has been reported that naloxone may be useful in the treatment of hypovolemic shock. However, the effects of naloxone on cardiac energy metabolism in acute hemorrhage have not been investigated. The effects of naloxone on myocardial metabolism were evaluated in the rat during acute hemorrhage with crystalloid resuscitation. Intramyocardial high energy phosphates, pyruvate, lactate and glycogen were measured. There was no significant difference in high energy phosphates, energy charge, lactate and glycogen contents between control, 1 mg naloxone and 10 mg naloxone groups. However, pyruvate level in hearts in the 10 mg naloxone group was significantly higher than that in control group. Therefore, naloxone reduced the lactate/pyruvate (L/P) ratio. Although naloxone did not improve the mitochondrial function, it improved the oxidation-reduction state in cardiac energy metabolism.

Enhancing effect of hydroxypropyl-beta-cyclodextrin on cutaneous penetration and activation of ethyl 4-biphenylyl acetate in hairless mouse skin.

The effect of hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) on the cutaneous penetration and activation of ethyl 4-biphenylyl acetate (EBA), a prodrug of non-steroidal anti-inflammatory drug 4-biphenylylacetic acid (BPAA), from hydrophilic ointment was investigated, using hairless mouse skin in vitro. When the hydrophilic ointment containing a complex of EBA with HP-beta-CyD was applied to the full-thickness skin, HP-beta-CyD facilitated the penetration of EBA into the skin, the conversion of EBA to BPAA in the epidermis and the transfer of BPAA to the receptor phase. Under the present condition, pre- and post-application of the ointment containing HP-beta-CyD onto the skin did not affect the cutaneous penetration of EBA and its activation. When the ointment containing the EBA:HP-beta-CyD complex was applied to the skin, the flux of BPAA through the tape-stripped skin was greater than that through the full-thickness skin, while the activation of the prodrug in the skin was slowed down by the tape-stripping. When propylene glycol was used as a vehicle, HP-beta-CyD no longer enhanced the cutaneous permeation of BPAA through the full-thickness skin. These results suggest that the enhancing effect of HP-beta-CyD on the cutaneous penetration of EBA would be ascribable largely to an increase in effective concentration of EBA in the ointment. Furthermore, the slow diffusion of EBA solubilized in HP-beta-CyD through the stratum corneum, together with the vehicle effect, could make the prodrug more susceptible to the metabolic process that is active in the epidermis, eventually leading to the facilitated activation of the prodrug.

Analysis of alcohol oxidase isozymes in gene-disrupted strains of methylotrophic yeast Pichia methanolica.

A cell-free extract of methanol-grown Pichia methanolica cells was found to contain nine alcohol oxidase (AOD) isozymes by active staining of a native polyacrylamide electrophoresis gel. Our previous study revealed that AOD in P. methanolica was encoded by two genes, MOD1 and MOD2, and the results of an experiment involving Candida boidinii as an expression host suggested that the AOD isozymes observed in P. methanolica were due to random association of Mod1p and Mod2p into an active octamer [Nakagawa et al., Yeast, 15, 1223-1230 (1999)]. This study was conducted using P. methanolica MOD1- and/or MOD2-gene disrupted strains to confirm a previous hypothesis. While the cell-free extract of the wild-type strain gave nine ladder bands, the mod1delta and mod2delta strains gave a single active AOD band corresponding to the mobilities of Mod2p and Mod1p on a native electrophoresis gel, respectively. The cell-free extract of glyceorl-grown wild-type cells gave a single band corresponding to Mod1p, showing that only MOD1 is expressed in glycerol-grown cells. While the expression of both MOD1 and MOD2 was induced by methanol, this finding and our previous observations indicated that the expression of MOD1 and MOD2 was controlled by a distinct regulatory mechanism in P. methanolica.

Progressive renal failure and blindness due to retinal hemorrhage after interferon therapy for hepatitis C virus-associated membranoproliferative glomerulonephritis.

We treated a 67-year-old Japanese woman with membranoproliferative glomerulonephritis (MPGN) and chronic active hepatitis associated with hepatitis C virus (HCV) infection. Treatment commenced with a daily dose of 6 MU IFN alpha-2b for 2 weeks, which was changed to three times weekly thereafter. After 2 weeks, HCV RNA in the serum was undetectable and there was a concomitant reduction in proteinuria. Treatment with IFN alpha-2b was discontinued because of severe headache and fever. Five weeks after the discontinuation of IFN alpha-2b, the patient experienced the sudden onset of visual loss due to retinal hemorrhage. Subsequently, proteinuria and renal function progressively deteriorated though HCV RNA was undetectable. This case exemplifies the need for careful monitoring of renal function and retinal lesions not only in patients receiving IFN but also in those following the discontinuation of IFN treatment.

A rare case of ectopic antidiuretic hormone-producing pancreatic adenocarcinoma: new diagnostic approach.

We describe a 73-year old man with the syndrome of inappropriate antidiuretic hormone secretion (SIADH) due to an ectopic ADH-producing pancreatic adenocarcinoma. His laboratory findings showed marked hyponatremia, and the water load test showed uncontrolled ADH secretion. The imaging studies revealed pancreatic body cancer. Histological examination revealed an adenocarcinoma of the pancreas, which was positive for ADH immuno-staining. The ADH in the tumor extract was 53.3 pg/g wet weight. In attempt to diagnose ADH-production from the tumor, the ADH in his pancreatic juice was measured and found to be 2.1 pg/ml. We conclude that it is valid to measure the ADH in pancreatic juice to diagnose ectopic ADH production by tumors.