Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

DNase I sensitivity of nuclear DNA measured by flow cytometry

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.     

Expression in Bacillus subtilis of the gene for human urogastrone using synthetic ribosome binding sites

Summary A chemically synthesised gene coding for human urogastrone which was earlier cloned in E. coli (Smith et al. 1982) has now been cloned into expression vectors for Bacillus subtilis Two types of constructs have been made, one giving production of methionylurogastrone and the other giving rise to a methionyl-urogastrone- galactosidase fusion polypeptide facilitating quantification of expression levels.The ribosome binding sites used in the expression plasmids are synthetically made oligonucleotides residing on short restriction fragments to allow easy replacement by other ribosome binding sites.Using shuttle vectors and constitutive promoters from Bacillus phages 105 and SPP1, we were able to detect levels of expression amounting to a few thousand molecules per cell during logarithmic growth in both E. coli and B. subtilis.     

Localization of glycoprotein glycosyltransferases in the Golgi apparatus of rat and mouse testis

Golgi fractions prepared from rat testis have been shown to be enriched in the following glycoprotein glycosyltransferases: N-acetylglucosaminyltransferase, 47-fold, galactosyltransferase, 33-fold, and N-acetylglucosaminide fucosyltransferase, 15-fold. Appreciably lower transferase levels were obtained in other subcellular fractions. In the mouse, Golgi fractions were prepared from testis homogenates, testis cell suspensions and partially purified testis germinal cells; these fractions were also enriched in the above glycoprotein glycosyltransferases. Electron microscopic analysis indicated that a major portion of the total transferase activity was located in the Golgi apparatus of both rat and mouse testis although these experiments could not rule out the possible presence of some transferase activity in other organelles.     

GENETIC DIVERSITY IN THE MONOECIOUS HYDROPHILE CERATOPHYLLUM (CERATOPHYLLACEAE)

This study surveys genetic variation in two clonal, monoecious, water-pollinated species that differ in their extent of sexuality and distributional range. Electrophoresis was used to quantify allozyme variability in 12 Wisconsin populations of the widespread Ceratophyllum demersum and the rare C. echinatum. Electrophoretic data indicate that populations of both species have low levels of sexual recombination, low levels of variation, and are structured genetically like inbreeding terrestrial plants. Ceratophyllum populations differ from “typical” clonal terrestrial plants by lower genetic diversity, lower proportions of multiclonal populations, and fewer genotypes per population. In two populations where sexual recombination is documented, heterozygosity is low with significant deficiencies. Monoecy in Ceratophyllum may be related to historical evolutionary factors, whereas vegetative reproduction has a greater influence on the genetic population structure of extant populations. The low genetic identity between C. demersum and C. echinatum supports their recognition as distinct species.     

Effects of Exogenous Androgen and Antiandrogen on Territorial and Nonterritorial Red-winged Blackbirds (Aves: Icterinae)

We investigated the role of testosterone (T) in territory establishment and maintenance in male red-winged blackbirds (Agelaius phoeniceus) by implanting exogenous T or pharmacological agents that block the action of T in nonterritorial floaters and territory owners. Floaters with artificially elevated plasma T levels were unable to obtain territories. Territory owners implanted with T did not expand their territories, although they engaged in more aggressive behavior than did control males. Flutamide, which binds to T receptors in target areas, had no effect on territoriality. However, most territory owners given a combination of flutamide and ATD, an aromatization blocker, lost portions of their territories even though they actively defended them. Our results 1) suggest that, although T influences aggressive behavior, elevated plasma levels alone are insufficient to overcome previously-established social relationships between territory owners or between owners and floaters; 2) indicate that impairing the action of T leads to reduced abilities of territory owners to maintain territories against vigorous challenges: and 3) support recent findings that T acts on reproductive behavior in birds through both androgenic and estrogenic metabolites.     

Diseases in North Sea fishes

Prior to the studies reviewed here, only lymphocystis and skeletal deformities of a variety of fish species and certain diseases of eel were known to occur in the German Bight (North Sea). From 1977 until now, 9 externally visible lesions on North Sea fishes were observed; in addition to those mentioned before, they comprise: fin rot, ulcerations, epidermal papilloma, hyperplasia, pseudobranchial tumour, eye diseases and gill swellings. With the exception of information on changes in frequencies of vertebral deformities of herring from the 1950's to the 1970's, there are no long-term data characterizing changes in frequencies of the diseases under study. For pseudobranchial tumours of cod and epidermal papilloma of dab, information is provided on occurrence and abundance. The distribution pattern of cod afflicted with pseudobranchial tumours is strongly influenced by the migratory behaviour of the fish. Epidermal papillomas of dab were more frequently found at stations within the inner German Bight than in neighbouring areas. The Bight is used for dumping of wastes from titaniumdioxide production. Further disease hot spots are areas off the Humber estuary and the British coast. Analysis of chromium in dab from the German Bight revealed elevated concentrations in epidermal tissues of specimens from the dumping area compared with that found in dab from neighbouring localities. Particulate iron was demonstrated to occur in mucous cells of dab from the dumping area. From increased levels of heavy metals with cancerogenic potential in sensitive target tissues and from increased prevalences of diseased fish in the dumping area it is concluded that these phenomena are possibly causally linked. In the vicinity of the Humber estuary high disease rates were encountered and areas with high prevalences of dab afflicted with epidermal papilloma extended over regions shown to be transport routes for persistent pollutants such as radioactive materials. It is therefore suggested that the long-range distribution of fish diseases in the southern North Sea might reflect the long-range transport of persistent pollutants.     

Kinetic Models of C3h Mouse Mammary Tumor Growth: Implications Regarding Tumor Cell Loss

Three models of tumor cell loss are described. the effects of cell loss on other cellular kinetic parameters are evaluated, and experiments which may distinguish among the models are discussed. Each model is based on a different cell-loss mechanism, and equations for the cell-cycle, cell-frequency distribution, the growth of both the proliferating and non-proliferating cell population, the growth fraction (GF), and the relative rate of volumetric growth, (dV/dt)/V, are derived. The following types of data are simulated for each model: the pulse labelling index, the mitotic index, and the labeling index as a function of time after a single or a series of ³H-TdR injections. the relative volumetric growth rate has the same mathematical form for each model. the PLM curves predicted by each model for the tumor lines studied (S102F and Slow) are not appreciably different. the predicted initial labeling index and mitotic index may differ significantly among the models depending upon the tumor line. the most striking difference among the models lies in the predictions regarding the labeling index as a function of time after a single or after a series of ³H-TdR injections. These types of labeling experiments should be valuable for distinguishing the different cell-loss mechanisms in solid tumors.