Biomedical subjects
Publications and source records attributed to W Heinen.
Extracranial aneurysms of the carotid artery in childhood are rare, and we found just nine cases described in the literature within the last 20 years. We report on the clinical appearance, diagnosis, and therapy of a carotid artery aneurysm in a 12-year-old girl and review the literature.
The reaction of iron sulfide (FeS) with H2S in water, in presence of CO2 under anaerobic conditions was found to yield H2 and a variety of organic sulfur compounds, mainly thiols and small amounts of CS2 and dimethyldisulfide. The same compounds were produced when H2S was replaced by HCl, in the H2S-generating system FeS/HCl/CO2. The identification of the products was confirmed by GC-MS analyses and the incorporation of H2 in the organic sulfur compounds was demonstrated by experiments in which all hydrogen compounds were replaced by deuterium compounds. Generation of H2 and the synthesis of thiols were both dependent upon the relative abundance of FeS and HCl or H2S, i.e. the FeS/HCl- or FeS/H2S-proportions. Whether thiols or CS2 were formed as the main products depended also on the FeS/HCl-ratio: All conditions which create a H2 deficiency were found to initiate a proportional increase in the amount of CS2. The quantities of H2 and thiols generated depended on temperature: the production of H2 was significantly accelerated from 50 degrees C onward and thiol synthesis above 75 degrees C. The yield of thiols increased with the amount of FeS and HCl (H2S), given a certain FeS/HCl-ratio and a surplus of CO2. A deficiency of CO2 results in lower thiol synthesis. The end product, pyrite (FeS2), was found to appear as a silvery granular layer floating on the aqueous surface. The identity of the thiols was confirmed by mass spectrometry, and the reduction of CO2 demonstrated by the determination of deuterium incorporation with DCl and D2O. The described reactions can principally proceed under the conditions comparable to those obtaining around submarine hydrothermal vents, or the global situation about 4 billion years ago, before the dawn of life, and could replace the need for a reducing atmosphere on the primitive earth.
Biofilm development in methanogenic fluidized-bed reactors with sand as the carrier was studied on a laboratory scale. The microorganisms present in consecutive layers of the biofilm of mature sludge granules were preliminarily characterized on the basis of their morphology, element composition and adhesion capacity and were compared to bacteria which take part in the initial colonization of sand. The early phase of biofilm development was monitored with reactors receiving waste-waters containing different mixtures of volatile fatty acids and inoculated with fluidized-bed reactor effluent for different lengths of time. The results obtained indicate that facultative anaerobic bacteria abundantly present in the outermost biofilm layers of mature sludge granules are probably the main primary colonizers of the sand. Methanothrix spp. or other methanogens were rarely observed among the primary colonizers. The course of biofilm formation was comparable under the various start-up conditions employed including variations in waste-water composition, inoculation and anaerobicity. However, omission of waste-water and thus of substrate resulted in rapid wash-out of the attached biomass.
The lipid content and fatty acid composition of a strain of Bacillus caldolyticus and of two facultative thermophiles (B. flavothermus and strain NZ-2) were analysed after growth at different temperatures. In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees. Changes of lipid content and protein/lipid ratio in B. caldolyticus between 60 degrees C and 100 degrees C and in strain NZ-2 between 45 degrees C and 70 degrees C were minor; in B. flavothermus the alterations in the 50 degrees C-70 degrees C range were more pronounced. The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids. The alterations of the fatty acid composition were most significant in B. caldolyticus, especially between 80 degrees C and 95 degrees C. In contrast, the main changes in B. flavothermus and NZ-2 were found to occur between 30 degrees C and 50 degrees C, and between 45 degrees C and 60 degrees C, respectively. Based on these data, strain NZ-2 could be characterized as the least and B. flavothermus as the most versatile of the three organisms.
The activity and stability of several enzymes from the facultative thermophile Bacillus flavothermus, grown within the mesophilic and thermophilic region at 34 degrees C, 43 degrees C, 52 degrees C and 70 degrees C, have been examined. While the temperature optima and maxima of all enzymes tested were found to remain unchanged at all growth temperatures, it was demonstrated that the heat stability of the proteins increased with ten perature, however, not uniformly for all enzymes. One exception was acetate kinase and the intrinsic stability of pyruvate kinase was found to increase only slightly. With all other proteins tested (alanine dehydrogenase, isocitric dehydrogenase and glucose-6-phosphate dehydrogenase, glutamate-oxalacetate and glutamate-pyruvate transaminase and myokinase) the intrinsic stability was found to increase to about 55 degrees C, but stayed unaltered at higher growth temperatures. Except for acetate kinase and myokinase, the enzymes could be stabilized by their respective substrates and the heat stability of the ES-complexes was found also to depend on the growth temperature of the cells. These data lead to the conclusion that the enzymes undergo a transition from heat-labile to thermostable within the growth temperature range between 44 degrees C and 51 degrees C while the thermal characteristics are not changed below and beyond this crucial region.
The thermostability of several enzymes from the facultative thermophilic actinomycete Streptomyces sp., derived from cells grown in the temperature range from 37 degrees C to 60 degrees C, has been examined. A correlation between the growth temperature of the cultures and the heat stability of the enzymes could be demonstrated for alanine dehydrogenase, isocitrate dehydrogenase, myokinase and pyruvate kinase. Except for the isocitrate dehydrogenase, which showed a linear increase of its stability throughout the entire temperature range, all enzymes exhibited a steep increase of the heat stability up to about 50 degrees C, but no further increase in the higher growth range, suggesting, that from 50 degrees C upward a shift to the exclusive production of thermostable protein occurs. Furthermore, the stability of alanine dehydrogenase and pyruvate kinase was found to increase substantially in presence of their substrates. In contrast, substrate-mediated stabilization was very weak with glucose-6-phosphate dehydrogenase and totally absent with acetate kinase, isocitrate dehydrogenase and myokinase. A comparison with previous observations with the same enzymes from Bacillus flavothermus showed, that enzymes from different organisms can have different thermal properties. Determination of the fatty acid composition of Streptomyces sp. cells, grown at different temperatures, showed relatively small alterations, with the main changes occurring between 37 degrees C and 40 degrees C.
A sample from a hot spring on the northern island of New Zealand contained five different thermophilic bacterial strains. One strain with peculiar properties, i.e. the formation of dark yellow colonies at 30 degrees C as well as at 70 degrees C, was further characterized. It was found to be a gram-positive, facultatively aerobic, motile Bacillus species, with terminal endospores. According to the physiologic properties the strain closely resembled B. coagulans. However, two typical characteristics were contradictory to this conclusion, namely the intense yellow pigmentation of the colonies and the range of growth temperature. The latter was found to reach from 40 to 70 degrees C, with an optimum at 60 degrees C under aerobic and at 65 degrees C under anaerobic conditions. Growth at moderate temperatures was slower than at 60 degrees C, but the final cell yields were almost equal. The strain can therefore be considered as facultatively thermophilic. The pigment, which was found to be located in the cytoplasmic membrane, was spectroscopically identified as a carotenoid. Because the characteristics of this strain did not correspond with any of the Bacillus species described thus far, we concluded, that we had isolated a novel strain, for which the name Bacillus flavothermus is proposed.
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When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, the responded to this situation primarily in the same way as phosphate-limited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate. Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite of hypophosphite. After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates. Adaptation to either PO3-3 or PO3-2 was completely abolished if the cells were again grown with PO3-3 as P-source, whereafter the entire process of adaptation had to be repeated. The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO3-3 and PO3-2, but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite. Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO3-4 to PO3-2. Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate.
Since the extracellular amylase from B. caldolyticus had been characterized as a Ca-dependent enzyme, we wanted to determine the function and specificity of this ion, and it's possible significance for the thermophilic properties of the enzyme. The first question concerned the substitution of Ca with regard to the activity at 70 C. Our data show that Sr or other divalent cations can only to a limited extent replace Ca, and that only in presence of Ca full activity can be achieved. When the majority of the binding sites of the subunits are occupied by cations other than Ca, an almost total loss of activity results. The reason for this kind of inactivation was found in the responsibility of Ca for the stability of the enzyme at high temperature. The omission of Ca then leads to an irreversible denaturation of the enzyme, so that after this kind of treatment no activity is detectable at either high or low temperature, which means that the amylase is not a thermostable enzyme in the classical sense. The stabilizing effect of Ca could not be substituted by any other of the cations tested. Experiments concerning the stability of the enzyme at various temperatures in absence of presence of Ca revealed that the enzyme is stable up to 55 C as long as trace amounts of Ca are available. If these are omitted by a chelator, the enzyme becomes unstable between 40 and 45 C. Experiments in which a certain protein concentration range was tested at 40 and 70 C with given concentrations of Ca showed that the stability, and with it the activity, at high temperature is directly related to the amount of Ca available: The more the Ca supply is limited, the less enzyme protein can be kept in the right configuration, and irreversibly denatures as a result. At low temperature, however, the enzyme becomes almost independent of Ca, and the small amounts necessary to obtain full activity can be replaced by other divalent cations. The main conclusions concerning the role of Ca are that at high temperatures it participates in achieving the correct configuration of the thermostable form of the enzyme, and that in this function it is irreplacable. At low temperature it acts as an unspecific cofactor. The other conclusion is that the amylase can exist either as a caldo-active, thermostable, Ca-dependent enzyme, or in a Ca-independent thermostable state with a lower activity.
One of the typical properties of the extracellular amylase produced by Bacillus caldolyticus is the tendency to disintegrate into subunits with a molecular weight (MW) of less than 10,000, when the enzyme is subjected to ultrafiltration. Disintegration is due to a loss of Ca-ions, leading to nonactive subunits. Activity can be fully restroed by addition of Ca-ions. Reactivation occurs also spontaneously if the low MW fraction is stored in glassware. Comparative ultrafiltration experiments with the subunit fraction with or without a supply of Ca revealed that in presence of this divalent cation the subunits reaggregated to the active enzyme. The different distribution patterns obtained in absence or presence of Ca showed that reactivation is directly linked to the formation of a high MW form of the protein. Substitution of Ca by other divalent cations also led to reaggregation. These aggregates are, however, inactive. The enzyme was found to be formed intracellularly in its low MW form. Experiments with protoplasts revealed that these are capable to produce and release the amylase. When the production of the enzyme by protoplasts declined, full restoration could be achieved by a recovery treatment. Normally, the enzyme released by the protoplasts consisted of equal portions of the high, medium, and low MW form of the amylase. If the cells were, however, depleted as a result of continued incubations, the extruded enzyme consisted increasingly of the low MW form, which could finally represent more than 80%. This trend could be completely reversed by the supply of carbon and nitrogen sources during the recovery treatment, whereafter the enzyme consisted again of the intially observed equal amounts of the three MW forms. Vesicles prepared from the protoplasts were also found to release amylase, but on a lower level, and only for a very limited time, with no possibility to regain activity by a recovery treatment. Subunit formation was also observed during column chromatography, which could be counteracted by a sufficient supply with Ca-ions.
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