Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice.

BACKGROUND: Heme oxygenase (HO)-1 is an enzyme that degrades heme to generate CO (a vasodilatory gas), iron, and the potent antioxidant bilirubin. A disease process characterized by decreases in vascular tone and increases in oxidative stress is endotoxic shock. Moreover, HO-1 is markedly induced in multiple organs after the administration of endotoxin (lipopolysaccharide [LPS]) to mice. METHODS AND RESULTS: To determine the role of HO-1 in endotoxemia, we administered LPS to mice that were wild-type (+/+), heterozygous (+/-), or homozygous null (-/-) for targeted disruption of HO-1. LPS produced a similar induction of HO-1 mRNA and protein in HO-1(+/+) and HO-1(+/-) mice, whereas HO-1(-/-) mice showed no HO-1 expression. Four hours after LPS, systolic blood pressure (SBP) decreased in all the groups. However, SBP was significantly higher in HO-1(-/-) mice (121+/-5 mm Hg) after 24 hours, compared with HO-1(+/+) (96+/-7 mm Hg) and HO-1(+/-) (89+/-13 mm Hg) mice. A sustained increase in endothelin-1 contributed to this SBP response. Even though SBP was higher, mortality was increased in HO-1(-/-) mice, and they exhibited hepatic and renal dysfunction that was not present in HO-1(+/+) and HO-1(+/-) mice. The end-organ damage and death in HO-1(-/-) mice was related to increased oxidative stress. CONCLUSIONS: These data suggest that the increased mortality during endotoxemia in HO-1(-/-) mice is related to increased oxidative stress and end-organ (renal and hepatic) damage, not to refractory hypotension.

Immobilized 4-aminophenyl 1-thio-beta-D-galactofuranoside as a matrix for affinity purification of an exo-beta-D-galactofuranosidase.

An alternative and fast method for the purification of an exo-beta-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-beta-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE-Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM D-galactono-1,4-lactone in a 100-500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-beta-D-galactofuranosidase was ascertained through binding to Concanavalin A-Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and Km and Vmax values of 0.311 mM and 17 mumol h-1 microgram-1 respectively, when 4-nitrophenyl beta-D-galactofuranoside was employed as the substrate.

Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET.

BACKGROUND: Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations. METHODS: We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx(R) amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS: Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx. CONCLUSIONS: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D-galactofuranosidase inhibitors.

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D-galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4-aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.

Pantothenate kinase activity in livers of genetically diabetic mice (db/db) and hormonally treated cultured rat hepatocytes.

Preparations from livers of fed and fasted genetically diabetic and nondiabetic mice (C57BL/KsJ db/db, db/+, or +/+) were used to determine whether changes in pantothenate kinase activity and/or properties corresponded to hormonally directed changes in liver total CoA content. Livers of fasted, nondiabetic mice had ratios of pantothenate kinase (PAK) to lactate dehydrogenase (LDH) activity 1.6 times values for fed nondiabetic controls, and they had a total CoA content per milligram of DNA that was 1.8 times control values. Livers of fed genetically diabetic mice had values for PAK/LDH and total CoA per milligram of DNA that were 1.5 and 2.8 times, respectively, those of nondiabetic controls. Liver PAK from genetically diabetic mice was inhibited by acetyl-CoA to the same extent as enzyme from nondiabetic mice and by CoASH to nearly the same extent. Rat hepatocytes in primary culture incubated with dibutyryl cAMP + theophylline + dexamethasone had PAK/LDH levels 1.5 times those of cells not treated with hormonal effectors, and PAK was inhibited to the same extent by acetyl-CoA and nearly the same extent by CoASH. The data show an increase in extractable hepatic PAK activity under conditions in which the total CoA content is elevated, and they suggest that glucocorticoids and cAMP levels contribute to the increased PAK activity.

Possible role of acetyl-CoA in the inhibition of CoA biosynthesis by ethanol in rats.

Ethanol, both administered to rats in vivo and added to cultured hepatocyte incubations, inhibits the conversion of [14C]pantothenate to coenzyme A (CoA). Data suggesting that the inhibition by ethanol involves its oxidation to acetate were obtained with rat hepatocytes maintained in primary culture. Ethanol, acetaldehyde and acetate were approximately equally effective inhibitors of [14C]pantothenate conversion to CoA (46-71%) and had no effect on uptake of [14C]pantothenate by hepatocytes. In the presence of saturating levels of acetate, acetaldehyde had no additional inhibitory effect. Cyanamide and diethyldithiocarbamate decreased the inhibition by acetaldehyde at the same concentration (10 microM), which saturated their ability to inhibit acetaldehyde oxidation. Studies with an isolated pantothenate kinase preparation showed that, of the ethanol metabolites, only acetyl-CoA was an effective inhibitor. Acetate and butyrate, which were both inhibitors of [14C]pantothenate conversion to CoA, increased the acetyl-CoA and decreased the free, unacylated CoA (CoASH) content of the cultured hepatocytes. The data were consistent with a mechanism for the inhibitory effect of ethanol that involves inhibition of pantothenate kinase by acetyl-CoA, but did not exclude a possible role of additional regulatory factors.

[The protrusion of thoracic intervertebral disc-thoracic spondylosis (author's transl)].

The protrusion of cervical intervertebral discs was divided into three pathological entities by Spurling; soft disc, hard disc and spondylosis. We applied these concept to the dorsal intervertebral disc disease and treated two cases of thoracic spondylosis. Case 1. A 41-year-old male entered the hospital because of the gradual progression of weakness of both legs of two months' duration. Since ten days before admission he had not had an errection and had not been to able to walk and micturate. He also complained of paresthesia radiating down the abdomen into both legs. There were no visceral complaints. Neurological examination revealed severe weakness of both legs with bilateral impairment of deep sensations and hypalgesia up to the level of T6. Reflexes in both legs were hyperactive with sustained clonus. Plantar responses were extensor bilaterally. Though plain X-rays showed no changes, tomography revealed a calcified intervertebral spur formation at the T5-6 interspace. A myelogram showed a complete block of the contrast medium at the level of the upper part of T6. The patient underwent a complete laminectomy from T3 through T6 and extradural anterior decompression with the removal of the calcified disc at the T5-6 interspace using an air drill. Postoperatively, he demonstrated an immediate improvement in sensation and a gradual recovery in motor power. At his follow-up examination 14 months after surgery he could walk without assistance. Case 2. A 47-year-old dwarfish woman (130 cm) with a low back pain and difficulty in walking for a few years duration was admitted. A few months before admission she felt pain at her left lateral abdomen. There was weakness of both legs, greater in the left. Reflexes in her left lower extremity were hyperactive with sustained clonus. Plantar responces were flexor bilaterally. Palin X-rays showed scoliosis of thoracic spine with the top at T7 level and calcified intervertebral masses at T10-11, T11-12 and T12-L1, extending into the canal that were confirmed more clearly by tomography. Myelography by a cisternal puncture disclosed a complete block at the level of T10. The patient underwent total laminectomy of T9 through L2 and extradural anterior decompression with the removal of calcified discs. At her follow-up examination 12 months after surgery she could walk for herself with some residual neurological signs, minimal weakness in the right leg and hypesthesia up to the level of T12 in the left. We have discussed the incidental, related diagnostic and operative problems of this disease.