[Galenic forms].
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Administration, Inhalation↗
Biomedical subjects
J Millet
Publications and source records attributed to J Millet.
[Drug therapy. General notions].
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Biological Availability↗
Cellulose Fermentation by an Asporogenous Mutant and an Ethanol-Tolerant Mutant of Clostridium thermocellum.
Two mutants of Clostridium thermocellum were isolated after UV light mutagenesis. Mutant A1, selected as asporogenous, exhibited a fermentation pattern similar to that of the wild type. However, at pH 6.5, the mutant degraded 12% more cellulose than did the wild type, leading to enhanced ethanol production. Mutant 647, selected as ethanol tolerant, was able to grow in medium containing 4% ethanol. During the early stage of the exponential growth phase, ethanol was produced as the main product, up to a concentration of about 9 g/liter. After 3 days of culture, 48.3 g (89% of the initial amount) of degraded cellulose per liter was fermented into 12.7 g of ethanol per liter.
Journal Article↗
Enhanced Cellulose Fermentation by an Asporogenous and Ethanol-Tolerant Mutant of Clostridium thermocellum.
A mutant of Clostridium thermocellum isolated after UV mutagenesis and selection for resistance to fluoropyruvate was found to be asporogenous and ethanol tolerant. The mutant was also an ethanol hyperproducer, able to ferment 63 g of cellulose into 14.5 g of ethanol per liter of medium. The ratio of ethanol to total organic acids produced by the mutant was increased, and H(2) production was decreased. Culture conditions were optimized for ethanol production by the new strain.
Journal Article↗
Evaluation of the effect of heparin and tetracycline on the cohesion of the dermal-epidermal junction.
In an attempt to demonstrate the effect of tetracycline or heparin therapy on the dermo-epidermal junction, the suction blister time was measured at the beginning and at the end of the study in 18 female Sprague-Dawley hairless rats, using the suction blister method. Suction blister time was defined as the time taken for the first sign of a vesicle to appear in the hole in the diaphragm of a suction chamber. 200 IU heparinate calcium was administered daily for 6 days in 6 rats. Six other rats received 27.5 mg tetracycline chlorhydrate daily for 6 days. The others did not receive any drug. Significant statistical differences in suction blister time before and after administration of the drugs were found in the heparin group (p less than 0.05), and in the tetracycline group (p less than 0.05), compared with the control group. Heparin and tetracycline were found to increase suction blister time significantly. These results suggest an increased cohesion in the dermo-epidermal junction due to heparin and tetracycline.
Animals↗
A new experimental model of venous thrombosis in rats involving partial stasis and slight endothelium alterations.
Venous thrombus formation is induced in rats using saline flushing in a venous bag created in the vena cava and subsequent stasis by stricture of the vein. Maximum thrombus weight is obtained in 15 minutes. Neither flushing nor partial stasis alone could trigger thrombus formation during this time. Transmission electron microscopy demonstrated that flushing induces discrete endothelial lesions with no evident desendothelialisation areas. Anticoagulant agents are found to be active in preventing thrombus formation. However, heparin or related compounds such as pentosan polysulphate are active at dose levels not demonstrating anticoagulant activity. Thrombosis could be induced in thrombocytopenic rats indicating the non-involving of platelets. Moreover, aspirin did not prevent thrombus formation. Ticlopidine, another antiplatelet agent, did show an activity which might not involved platelets. This model thus possesses the features necessary for reliable investigation of potential antithrombotic agents for human use.
Animals↗
The cloned cel (cellulose degradation) genes of Clostridium thermocellum and their products.
More than 10 cel genes of Clostridium thermocellum were cloned and expressed in Escherichia coli. Four of them, celA, celB, celC and celD, were studied in detail. The corresponding endoglucanases were purified from E. coli and characterized. The over-produced endoglucanase D, purified from cytoplasmic granules, was crystallized.
Amino Acid Sequence↗
The endogenous cytochemically assayable Na-K-ATPase inhibitor and its relation to hypertension.
Acute volume expansion endows the plasma with the capacity to cause a natriuresis, to inhibit Na-K-ATPase and to stimulate vascular reactivity. These changes are due to a change in the concentration of two or more substances in the plasma. Atrial natriuretic peptides do not inhibit Na-K-ATPase and they decrease vascular reactivity. The search for the Na-K-ATPase inhibitor has been hampered by the lack of specificity of most assays which demonstrate the presence of many irrelevant Na-K-ATPase inhibitors. Cytochemical bioassays, however, appear to have a certain selectivity. They disclose the presence of a Na-K-ATPase inhibitor in the plasma, the concentration of which is controlled by salt intake but the only known substance so far tested, which these bioassays will detect, is ouabain. The hypothalamus contains the highest concentration of the cytochemically bioassayable inhibitory material and its content is also controlled by salt intake. The substance responsible for the cytochemically bioassayable inhibitory activity in the plasma and the hypothalamus share the same physico-chemical properties and on extraction appear in the same fraction. It is possible therefore, that the Na-K-ATPase inhibitor which is detectable by the cytochemical assays in the plasma and hypothalamus is the same. The concentration of cytochemically bioassayable Na-K-ATPase inhibitory activity is raised in the plasma of patients with essential hypertension, the SHR rat and the Milan hypertensive rat, as is the concentration of atrial natriuretic peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum.
Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.
Cellulase↗
Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli.
The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.
Carboxymethylcellulose Sodium↗
Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease.
A colony screening procedure was devised to detect Bacillus subtilis mutants containing temperature-sensitive trypsin-like intracellular protease activity. The enzyme was characterized as a non-sulfhydryl serine protease on the basis of inhibitor studies. It was also inhibited by D- or L-histidine but not by any other amino acid tested. The long-term survival at 45 degrees C of these mutants in a minimal salts medium was decreased, with rapid lysis occurring within 24 h. A D-histidine function in long-term survival and inhibition accounted for the presence of additional protease mutants among survivors of histidine auxotrophs selected for their ability to utilize D-histidine. In addition to being lysed when incubated at 45 degrees C under nongrowth conditions, all of the protease mutants had a decreased rate of protein turnover and produced spores deficient in a major low-molecular-weight spore coat polypeptide. The morphology of the undercoat layers was altered, but there was no effect on spore heat resistance or on germination. The missing spore coat polypeptide appeared to be processed from a larger precursor by cleavage to produce N-terminal histidine. A defect in this protease could account for the lack of processing and thus the absence of this polypeptide in spore coats.
Bacillus subtilis↗
Ultrastructure and extreme heat resistance of spores from thermophilic Clostridium species.
The heat resistance and ultrastructural features of spore suspensions prepared from Clostridium thermocellum LQRI, Clostridium thermosulfurogenes 4B, and Clostridium thermohydrosulfuricum 39E were compared as a function of decimal reduction time. The decimal reduction times at 121 degrees C for strains LQRI, 4B, and 39E were 0.5, 2.5, and 11 min. The higher degree of spore heat resistance was associated with a spore architecture displaying a thicker cortex layer. Heat resistance of these spores was proportional to the ratio of spore cortex volume to cytoplasmic volume. These ratios for spores of strains LQRI, 4B, and 39E were 1.4, 1.6, and 6.6, respectively. The extreme heat resistance and autoclavable nature of C. thermohydrosulfuricum spores under routine sterilization procedures is suggested as a common cause of laboratory contamination with pure cultures of thermophilic, saccharide-fermenting anaerobes.
Clostridium↗
Purification and properties of an endo-beta-1,4-glucanase from Clostridium thermocellum.
The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate which, until now, has not been resolved into individual enzyme components. By using QAE-Sephadex A50 chromatography in the presence of 6 M urea, it was possible to split the complex into distinct protein fractions. One of these fractions contained an endo-beta-1,4-glucanase which was isolated at a high degree of purity and was identified by its ability to hydrolyze trinitrophenylated carboxymethylcellulose. The enzyme is of monomeric nature, with a molecular weight of 56,000. It has an isoelectric pH of 6.2 and an optimum pH of 6.0. It hydrolyzed carboxymethylcellulose and, at a slower rate, cellulose powder. The major end products of cellulose degradation are glucose, cellobiose and cellotriose; cellotetrose is formed as an intermediate product. No specific small molecular weight activator or inhibitor was found except cellobiose and, to a lesser extent, glucose, which at high concentrations partially inhibit the activity of the enzyme. The temperature dependence of the enzyme is related to the thermophilic character of the producing microorganism.
Carboxymethylcellulose Sodium↗
Characterization of an inhibitor of the intracellular protease from Bacillus subtilis.
A specific inhibitor of intracellular serylprotease from Bacillus subtilis has been isolated from both growing and sporulating cells. Like other protease inhibitors isolated from eukaryotic cells, the inhibitor from B. subtilis is a thermostable protein. A purification method is described. The molecular weight estimated by Biogel filtration and SDS gel electrophoresis is about 15,500. Both proteolytic and esterolytic activities of intracellular protease are equally sensitive to inhibition. With azocoll or Z-tyrosine p-nitrophenylester as substrates, noncompetitive inhibition patterns are observed. The inhibitor has no effect on the proteolytic or esterolytic activities of the extracellular serylprotease. A similar thermostable inhibitor is also present in Bacillus megaterium.
Bacillus megaterium↗
Characterization of a protein inhibitor of intracellular protease from Bacillus subtilis.
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Bacillus subtilis↗
[Thermosensitive sporulation and extracellular serylprotease mutant of B. subtilis].
Isolation and properties of B. subtilis ts 19 mutant, isolated as thermosensitive for sporulation, are described. At the non permissive temperature (42degreesC), the mutant cells are blocked at stage zero of sporulation and do not excrete extracellular enzymes such as serylprotease and esterase. At the permissive temperature (30degreesC), sporulation and excretion of extracellular enzymes are normal but the serylprotease is modified in its structure. Two molecular forms of this enzyme can be separated by polyacrylamide elecctrophoresis, both more thermolabile than the corresponding enzyme of the mother strain. Experiments of reversion and of transformation for the sporulation character have suggested that ts 19 contained two independent thermosensitive mutations. One of them is responsible for the pleiotropic Spo OA phenotype at the non permissive temperature. The other mutation is likely to reside in the structural gene coding for the extracellular serylprotease and leads to the formation of a modified enzyme which hydrolyzes itself into at least two types of more stable molecules. No conclusion can be drawn with certainty concerning the physiologi-al role of the extracellular serylprotease in sporulation. It may be pointed out however that transformants for the Sp+ character at 42degreesC keep the same impaired serylprotease as the ts 19 mutant and sporulate, at any temperature, as well as the wild strain.
Bacillus subtilis↗
[Purification and properties of an esterase excreted during sporulation of Bacillus subtilis].
After growth in Difco Nutrient Broth, several proteolytic enzymes are excreted by B. subtilis, Marburg strain, namely a metalloprotease and a seryl protease. We report here the purification and some biochemical properties of a third extracellular hydrolytic enzyme for which we propose the term of esterase. As the serylprotease, the esterase is a seryl enzyme endowed with both proteolytic and esterolytic activities. Nevertheless the esterase differs from serylprotease in many aspects. In particular, it is an acidic enzyme with a low proteolytic activity and a high esterolytic activity. Its specificity toward synthetic substrates is restricted. The enzyme is active only on esters of amino acids and particularly on those of tyrosine. With Bz Tyr O Et as substrate the esterase displays a maximum of activity between pH 7.2 and 8.1 with a Km of 1.3.10-minus 3 M at 30 degrees C. At the end of this work, two questions remain unanswered: 1) the origin of the multiplicity of the bands revealed by polyacrylamide electrophoresis in the purest fraction; 2) the nature of the physiological substrate of the enzyme.
2,2'-Dipyridyl↗