Tn 5 to assess soil fate of genetically marked bacteria: screening for aminoglycoside-resistance advantage and labelling specificity

Abstract The reliability of Tn 5 as labelling tool was investigated in soil microcosm. The occurence of a selective in soil microcosm. The occurence of resistances encoded by Tn 5 nptII gene was assesed by kanamycin and neomycin amendment. The bioassay developed to monitor the persistence of the soil-added kanamycin did not detect the antibiotic activity in soil extract. A nptII -engineered Escherichia coli strain showed no enhanced survival in aminoglycoside amended soil. Tn 5-marker properties were investigated within indigenous bacteria to determine the specificity of labelling to follow the fate of recombinant DNA. Kanamycin and neomycin resistant population levels made Tn 5 aminoglycoside-resistance phenotype non-sensitive enough to select a soil dissemination of the labelled DNA. The unexpected occurrence of homologous sequences among soil organisms also prevented Tn 5 from being a specific DNA marker. By contrast, colony hybridization did not reveal homology to nptII suggesting its use as a reliable gene transfer indicator.     

The nervous system of the tube-worm Chaetopterus variopedatus (Polychaeta)

Silver impregnation of serial histological sections of the tubeworm Chaetopterus variopedatus revealed the presence of a subepidermal nervous system. The anterior nervous system is delimited by the first 11 segments and comprises (1) two dorsolateral cerebral ganglia and lateral instead of ventral nerve cords which are widely separated and thus connected by unusually long commissures, (2) a pharyngeal ganglion in the fourth segment which is connected to the cerebral ganglia by pharyngeal nerves and constitutes along with the pharyngeal plexus a stomatogastric or enteric nervous system, and (3) small, presumably segmental ganglionic swellings along the lateral nerve cords from which emerge commissures and parapodial nerves. No subesophageal ganglion or periesophageal connective could be identified. The lateral nerve cords converge toward the midline in the 12th segment to form the posterior nervous system comprising a pair of ventromedian nerve cords with their repetitive segmental ganglia from which emerge numerous short commissures and three segmental nerves coursing toward the dorsal and ventral regions of parapods and toward the neuropod. Light and electron microscopic investigations of cerebral and segmental ganglia showed an arrangement of inner neuropile and of unipolar neuron somata at the periphery. The neuropile comprises numerous neurites ranging in diameter from 0.5 to 10 μm and making polarized or symmetrical synaptic junctions with each other. The pharyngeal ganglion consists of a similar neuropile and of a large mass of cell bodies which is traversed by an elaborate network of sinuses and harbors three types of neurosecretory cells in addition to the conventional neuron somata. These findings are interpreted in the framework of the highly specialized morphological features and habits of Chaetopterus, and the welldeveloped stomatogastric system is considered to be related to control of the feeding activities.     

The relationship between cell volume,ploidy, and functional activity in differentiating hepatocytes

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radio-immunoassay. The capacity of this cell type to store³⁵S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up^99mTC-colloid sulfur and to incorporate⁵⁹Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.     

α,β-dicarbonyl reduction by Saccharomycesd-arabinose dehydrogenase

An α,β-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme d-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive α,β-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent K_m values of ∼ 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s^− 1 at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP⁺ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of α,β-dicarbonyl substrates was further supported by the observation that ara1-Δ knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.