Methane production from acetamide in an upflow anaerobic sludge-blanket reactor based on a synergistic association between an aerobic rod and methanogens

Acetamide degradation was investigated in a bench-scale upflow anaerobic sludge-blanket (UASB) reactor, successively fed with acetamide, acetate and acetamide, over a period of 343 days, at different hydraulic retention times (t _HR). The reactor was seeded with the sludge previously described [Guyot et al. (1994) Appl Microbiol Biotechnol, 42:452-456], in which methanogenesis from acetamide was performed through a synergistic relationship between an acetamide-degrading, aerobic rod and methanogens. When the reactor was fed acetamide, the chemical oxygen demand (COD) removal efficiency was 86% at volumetric loads less than 1.18 kg COD m^–3 day ^–1. At higher volumetric loads, the efficiency decreased markedly, e.g. 50.9% at a volumetric organic load of 3.39 kg COD m^–3 day^–1 (1 day t _HR) with an accumulation of both acetamide and acetate. The same reactor, when fed with acetate at t _HR 1 day, reached a high COD removal (99%). Evidence of the inhibition of acetate degradation by acetamide is presented. After a long period (135 days) without feeding the reactor with acetamide, the sludge reactor was still capable of degrading acetamide when this substrate was supplied again. It seems that the synergistic degradation of acetamide by aerobes and methanogens present in the UASB reactor sludge is stable over a long period (343 days), in spite of limiting concentrations of dissolved oxygen in the feed.     

The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis . Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC–lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90°C, whereas B. subtilis AhrC was largely inactivated at 65°C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85°C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus . This pronounced resistance of the repressor–operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.     

The stationary state of epithelia

A tissue is a geometrical, space-filling, random cellular network; it remains in this steady state while individual cells divide. Cell division is a local, elementary topological transformation which establishes statistical equilibrium of the structure. We describe the physical conditions to maintain stationary the epidermis (of mammals or of the cucumber), in spite of the fact that cells constantly divide and die. Specifically, we study the statistical equilibrium of the basal layer, a corrugated surface filled with cells, constituting a two-dimensional topological froth. Cells divide and detach from the basal layer, and these two topological transformations are responsible for the stationary state of the epidermis. The topological froth is capable of responding rapidly and locally to external constraints, and is a good illustration of the plasticity of random cellular networks.Statistical equilibrium is controlled by entropy, both as a measure of disorder and as information, and is characterized by observable relations between average cell shapes and sizes. The technique can be applied to any random cellular network in dynamical equilibrium. Mitosis as the dominating topological transformation and the fact that the distribution of cell shapes is very narrow are the only inputs specific to biology.

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Resume Un tissu est, à première vue, un pavage aléatoire d'une surface ou d'un volume par des polygones (polyèdres) topologiques, les cellules. Ce pavage reste dans un état stationnaire alors que les cellules se divisent constamment. Nous décrivons les conditions physiques nécessaires à l'état stationnaire de l'épiderme (des mammifères et du concombre), en dépit du fait que ses cellules se divisent et meurent. En particulier, nous étudions l'équilibre statistique de la couche basale, une surface couverte de cellules constituant une mousse topologique aléatoire. Les cellules se divisent et se détachent de la couche basale, et ces transformations topologiques sont responsable de l'état stationnaire de l'épiderme. Cette mousse topologique est capable de répondre rapidement et localement à des contraintes externes. C'est un exemple de plasticité de structures cellulaires aléatoires.L'équilibre statistique est contrôlé par l'entropie qui est ici à la fois une mesure du désordre et une quantité d'information. Il est caractérisé par des relations facilement observables entre les formes des cellules et leurs dimensions. Les seuls éléments spécifiques aux tissus biologiques sont la mitose comme transformation topologique dominante, et l'étroitesse de la distribution des formes de cellules.

     

Posterior dental size reduction in hominids: The Atapuerca evidence

In order to reassess previous hypotheses concerning dental size reduction of the posterior teeth during Pleistocene human evolution, current fossil dental evidence is examined. This evidence includes the large sample of hominid teeth found in recent excavations (1984–1993) in the Sima de los Huesos Middle Pleistocene cave site of the Sierra de Atapuerca (Burgos, Spain). The lower fourth premolars and molars of the Atapuerca hominids, probably older than 300 Kyr, have dimensions similar to those of modern humans. Further, these hominids share the derived state of other features of the posterior teeth with modern humans, such as a similar relative molar size and frequent absence of the hypoconulid, thus suggesting a possible case of parallelism. We believe that dietary changes allowed size reduction of the posterior teeth during the Middle Pleistocene, and the present evidence suggests that the selective pressures that operated on the size variability of these teeth were less restrictive than what is assumed by previous models of dental reduction. Thus, the causal relationship between tooth size decrease and changes in food-preparation techniques during the Pleistocene should be reconsidered. Moreover, the present evidence indicates that the differential reduction of the molars cannot be explained in terms of restriction of available growth space. The molar crown area measurements of a modern human sample were also investigated. The results of this study, as well as previous similar analyses, suggest that a decrease of the rate of cell proliferation, which affected the later-forming crown regions to a greater extent, may be the biological process responsible for the general and differential dental size reduction that occurred during human evolution. © 1995 Wiley-Liss, Inc.     

In vitro colonization ability of human colon mucosa by exogenous Lactobacillus strains

Abstract A 5.4 kb Hind III DNA fragment carrying the gene encoding raw starch-digesting α-amylase (RSDA), has been previously cloned from Bacillus circulans F-2 and expressed in Escherichia coli [Kim et al. (1990) Biochim. Biophys. Acta 1048, 2233–2238]. Interestingly, when the cell extract of E. coli harboring a plasmid carrying this fragment was incubated with l M NaCl, it exhibited about 10 times higher enzyme activity than when assayed without NaCl. Differential zymograms showed two different amylase activities: one for RSDA and the other for a salt-dependent a-amylase (SDA). Even though RSDA activity was detected without NaCl, SDA activity was detected only in high concentrations of NaCl. SDA activity was fully detected at above l M NaCl. Results from subcloning of the genes, fractionation analysis of cell extracts, and immunological assays clearly suggested that the two amylases are genetically distinct and that genes for both enzymes are closely linked on the 5.4 kb DNA fragment.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

Nonsense RNA: a tool for specifically inhibiting the expression of a gene in vivo

We describe a general technique to inhibit gene expression in eukaryotic cells. The gene we chose to inhibit was the E. coli LacZ gene (encoding beta-galactosidase), which has previously been cloned into a eukaryotic expression vector [1]. This plasmid is called pCH110. We constructed a variant of pCH110 in which we flipped a 2566 base pair 5' fragment of the LacZ gene into the antiparallel orientation. The plasmid containing this mutated LacZ gene is called pNSLacZ (NS signifies non-sense coding sequence). When equal amounts of pCH110 and pNSLacZ are co-transfected into 3T6 mouse fibroblasts, the beta-galactosidase activity is decreased by approximately a factor of ten. Increasing the ratio of pNSLacZ to pCH110 above 1:1 does not appreciably increase the level of inhibition. Next, we prove the specificity of the inhibition by adding a third gene to the transfection mixture. For this purpose, we used pSVneo beta, a plasmid which expresses a phosphotransferase. We found that even when the beta-galactosidase activity was diminished by a factor of 10, the phosphotransferase activity was unaffected. Therefore, we have demonstrated that: the presence of an antiparallel copy of the LacZ gene results in a significant and specific diminution of the LacZ gene's expression; only a fraction of the LacZ gene needs to be in the antiparallel orientation in order to observe this effect. These results suggest that this technique can serve as a tool to decrease the level of gene expression in order to study the function of specific genes, or as a therapeutic manoeuvre in the treatment of disorders of abnormal gene expression.