Cell-surface enrichment of fucosylated glycoconjugates in the 8- to 16-cell mouse embryo

Summary The destination of ³H-fucose was examined in preimplantation mouse embryos at the 8- to 16-cell stage by autoradiography of semi-thin sections. At the end of a 16–18 h incubation in medium containing 200 Ci/ml L-[6-³H] fucose the density of silver grains over the combined surface of cells where they faced their neighbours was 40% greater than that over the outer polar cell surface and 80% greater than that over the cytoplasm. These differences were statistically significant (p<0.01). The outer cell surface also had a grain density above that found over the cytoplasm (p<0.01). There was a rapid decrease of 18–24% in the incorporated radioactivity during the first 1.5 to 3 h in chase medium containing 10 mM cold fucose. Following this the grain density remained relatively stable until it decreased further between 24 and 48 h. By 48 h there was no significant difference between the density of grains over cytoplasm, apposed cell surfaces and outer cell surface: all were reduced compared to the initial values, to 29% in the case of the surfaces and 10% in the case of the cytoplasm. These results indicate that there is an enrichment in the incorporation of ³H-fucose into macromolecules at the cell surfaces where neighbouring cells confront one another relative to the outer polar cell surface, as well as the cytoplasm, at the 8- to 16-cell stage. Conclusions about the relative density of fucoconjugates per unit length of apposed and polar membrane cannot be reached from such a light microscopic study. Fucoconjugates appear to belong to two groups: the first are turned over relatively rapidly (within 3 h after removal from radioactive medium) while the second are more stable, decaying between 24 and 48 h after removal from ³H-fucose.     

The viscostat: Productstat method of feed-rate control in continuous fermentations

Xanthan biopolymer has been produced in a single-stage continuous fermentation with Xanthomonas campestirs NRRL B-1459, using a viscostal control method instead of the conventional chemostat method. A Bendix Ultraviscoson

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sensed the fermentor viscosity, and the recorder–controller actuated the feed medium pump in an on–off control mode. Since all continuous fermentations eventually become contaminated or suffer culture variation, this work served also to demonstrate the effectiveness of the viscostat control. Neither the presence of a mold contaminant with specific growth rates lower than that of X. campestris, nor the presence of a bacterial contaminant of specific growth rate greater than X. campestris, affected the maintenance of constant viscosity in this control system.     

Klebsielleae in drinking water emanating from redwood tanks.

A survey was made of the bacteriological quality and chlorine content of 33 public and private water systems that utilize redwood storage tanks. Coliforms of the genera Klebsiella and Enterobacter were isolated from 9 of 10 private drinking water systems and from 11 of 23 water systems in state and federal parks. Total coliform counts in the private systems exceeded federal membrane filter guidelines by as much as 10-to 40-fold. Coliform counts were highest in the newer reservoirs. Factors contributing to poor water quality are: lack of automated chlorination equipment or an insufficient supply to maintain a residual, common inlet/outlet plumbing design, and lengthy average retention periods. The latter two factors contribute to improper mixing and stagnation of the water, whereas the former allows microbes to multiply on the water-soluble nutrients that leach from the wood. Wooden reservoirs exert a high chlorine demand, and 0.4 ppm of chlorine residual in the incoming tank water proves inadequate. It is suggested that specific water-soluble nutrients in redwood (and in numerous other types of botanical material) induce a natural nutritional selection for coliforms of the tribe Klebsielleae.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

Functional variation among frugivorous birds: implications for rainforest seed dispersal in a fragmented subtropical landscape

Seed dispersal plays a critical role in rainforest regeneration patterns, hence loss of avian seed dispersers in fragmented landscapes may disrupt forest regeneration dynamics. To predict whether or not a plant will be dispersed in fragmented forests, it is necessary to have information about frugivorous bird distribution and dietary composition. However, specific dietary information for frugivorous birds is often limited. In such cases, information on the seed-crushing behaviour, gape width and relative dietary dominance by fruit may be used to describe functional groups of bird species with respect to their potential to disperse similar seeds. We used this information to assess differences in the seed dispersal potential of frugivorous bird assemblages in a fragmented rainforest landscape of southeast Queensland, Australia. The relative abundance of frugivorous birds was surveyed in extensive, remnant and regrowth rainforest sites (16 replicates of each). Large-gaped birds with mixed diets and medium-gaped birds with fruit-dominated diets were usually less abundant in remnants and regrowth than in continuous forest. Small-gaped birds with mixed diets and birds with fruit as a minor dietary component were most abundant in regrowth. We recorded a similar number of seed-crushing birds and large-gaped birds with fruit-dominated diets across site types. Bird species that may have the greatest potential to disperse a large volume and wide variety of plants, including large-seeded plants, tended to be less abundant outside of extensive forests, although one species, the figbird Sphecotheres viridis, was much more abundant in these areas. The results suggest that the dispersal of certain plant taxa would be limited in this fragmented landscape, although the potential for the dispersal of large-seeded plants may remain, despite the loss of several large-gaped disperser species.     

Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake

Intermedin (IMD)/adrenomedullin-2 (AM2) is a novel peptide that was independently discovered by two groups. The 47-amino acid peptide is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. IMD should therefore have actions similar to those of AM and CGRP. Indeed, like AM and CGRP, intravenous administration of IMD decreased blood pressure in rats and mice. We demonstrate here that immunoreactive IMD is present in plasma as well as heart, lung, stomach, kidney, pituitary, and brain. Because IMD is present in brain and both AM and CGRP have potent central nervous system (CNS) effects, we examined the ability of IMD within brain to regulate blood pressure and ingestive behaviors. Administration of IMD into the lateral cerebroventricle of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by intravenous administration of phentolamine, indicating that IMD activates the sympathetic nervous system. Intracerebroventricular administration of IMD also inhibited food and water intake in sated and in food- and water-restricted animals. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. Our data indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both AM and CGRP receptors.     

Inhibition of early and late events of the HIV-1 replication cycle by cytoplasmic Fab intrabodies against the matrix protein, p17.

BACKGROUND: The HIV-1 matrix (MA) protein, p17, contains two subcellular localization signals that facilitate both nuclear import of the viral preintegration complex early during infection and virus particle assembly late in infection. The dual role of MA in both the afferent and efferent arms of the HIV-1 life cycle makes it an important target for intracellular immunization-based gene therapy strategies. MATERIALS AND METHODS: Here we report, using a new bicistronic vector, that an intracellular Fab antibody, or Fab intrabody, directed against a carboxy-terminal epitope of MA from the Clade B HIV-1 genotype, can inhibit HIV-1 infection when expressed in the cytoplasm of actively dividing CD4+ T cells. RESULTS: Marked inhibition of proviral gene expression occurred when single-round HIV-1 CAT virus was used for infections. In challenge experiments using both laboratory strains and syncytium-inducing primary isolates of HIV-1, a substantial reduction in the infectivity of virions released from the cells was also observed. CONCLUSIONS: This novel strategy of simultaneously blocking early and late events of the HIV-1 life cycle may prove useful in clinical gene therapy approaches for the treatment of HIV-1 infection and AIDS, particularly when combined with genetic or pharmacologic-based strategies that inhibit other HIV-1 target molecules simultaneously.     

Evolution of the Sry genes

Existing DNA sequence data on the Sry gene, the mammalian sex- determining locus in the Y chromosome, were analyzed for primates, rodents, and bovids. In all three taxonomic groups, the terminal sequences evolved faster than the HMG (high mobility group) boxes, and this applies both to synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions. Similar intragenic correlation between synonymous and nonsynonymous substitution rates was not found either in other mammalian genes that contain a conservative box (Sox, Msx) or in the MADS-box genes of plants. The rate of nonsynonymous substitutions exceeds significantly that of synonymous substitutions in the terminal Sry sequences of apes. We did not find good support for the hypothesis that the high evolutionary rate of Sry would be associated with a promiscuous mating system.      

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Biofiltration for the removal and ‘detoxification’ of a complex mixture of volatile organic compounds

A study was performed to determine the effectiveness of using biofiltration for the removal of a complex mixture of volatile organic compounds (VOCs) air-stripped from petroleum hydrocarbons. A biofilter was constructed which contained 264 cm³ of packing material (Celite^? R-635). The unit was inoculated with a mixed culture containing a hydrocarbon-degrading Pseudomonas sp and an Alcaligenes sp. Several of the major compounds in the VOC mixture were monitored individually, along with the total VOCs, using gas chromatography. The average influent concentration of the VOC mixture was 320 ppmv and the average total VOC removal rate was over 56%, with the average removal rate of the monitored individual compounds ranging from 49–90%. After 30 days of operation the average overall removal rate was 69% and the removal of the major compounds averaged 92%. The toxicity and mutagenicity of the air stream was monitored using the Microtox and Ames assays, respectively. These data show marked decreases in toxicity and mutagenicity of the air stream as a result of the biofiltration treatment. The biofiltration system, therefore, was not only effective in removing VOCs from the air stream over an extended time-period, but was also effective in greatly reducing the toxicity and mutagenicity associated with the remaining VOCs. Received 03 July 1997/ Accepted in revised form 25 November 1997