Bacterial productivity in the water column and sediments of the Georgia (USA) coastal zone: Estimates via direct counting and parallel measurement of thymidine incorporation

Three methods of estimating bacterial productivity were compared using parallel samples of Atlantic Ocean water (within 0.25–15 km of the Georgia coast). The frequency-of-dividing cells (FDC) method and the [³H]thymidine incorporation method gave results which were strongly correlated (r=0.97), but the FDC estimates were always higher (X2 to X7) than the [³H]thymidine estimates. Estimates of bacterial productivity ranged from 2–4×10⁸ cells·l^–1·h^–1 at 0.25 km from shore to 1–9×10⁷cells·l^–1·h^–1 at 15 km. A method involving incubation of 3-m filtrates and direct counting gave results that could not be easily translated into estimates of bacterial productivity. Application of the FDC method to sediment samples gave high productivity estimates, which could be not reconciled with productivity estimates based on sediment oxygen uptake.     

Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B)

Hirschsprung disease is a congenital malformation affecting 1 in 5000 live births. The absence of parasympathetic neuronal ganglia (Meissner, Auerbach) in the hindgut results in poor coordination of peristaltic movement, and a varying degree of constipation. Four different genes have been implicated in the pathogenesis of Hirschsprung disease: the RET tyrosine kinase receptor gene; one of its ligands, the glial cell line-derived neurotrophic factor (GDNF) gene; the endothelin receptor B (EDNRB) gene; and its ligand, endothelin-3 (EDN3). Recently, combinations of mutations in two of these genes (RET and GDNF) have been reported in Hirschsprung patients. We report a family with missense mutations in both the RET gene (R982C) and the EDNRB gene (G57S). In this family, three out of five members have the two mutations, but only one, a boy, has the Hirschsprung disease phenotype. This illustrates the complexity of the molecular background of Hirschsprung disease. Received: 23 January 1998 / Accepted: 24 March 1998     

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Seed coat variation in Glycine Willd. subgenus Glycine (Leguminosae) by SEM

Seed of the genusGlycine Willd. typically exhibits a muriculate appearance resulting from adherence to the true seed coat of the perisperm or inner pod wall layer. Thickened cell walls of the perisperm superimpose a reticulate network on the seed coat, the type of network ranging from alveolate to stellate depending on the shape of the perisperm cells. Tubercles distributed at intervals give the seed its roughened appearance. Seed lacking an attached perisperm appears smooth and shiny. Seed morphology of 62 collections representing the six species of the subgenusGlycine is examined in detail to elucidate inter-and intraspecific variability. Seed perisperm pattern appears to be characteristic for each species, but there are exceptions.Glycine canescens F. J. Herrn. andG. clandestina Willd. seeds possess a reticulate network and tubercles of irregular shape, the perisperm appearing granular inG. clandestina. Seeds ofG. latrobeana (Meissn.) Benth. andG. tabacina (Labill.) Benth. lack a distinct network and have stellate tubercles; the perisperm is granular inG. latrobeana and some plants ofG. tabacina. A few collections ofG. clandestina approachG. tabacina in seed appearance.Glycine tomentella Hayata seeds exhibit a regularly alveolate arrangement, while those ofG. falcata Benth. lack a perisperm layer altogether. Variation in seed coat within a species can usually be linked to differences in chromosome number or some aspect of gross morphology. Diploid collections ofG. tomentella (2n = 40) exhibit recognizable differences in seed morphology compared with tetraploids (2n = 80), coincident with other striking dissimilarities in gross morphology. An incompletely attached perisperm is accompanied by aneuploidy in severalG. tomentella accessions, while other 78 and 38 chromosome aneuploids produce normal seeds.     

Mutants of Salmonella typhimurium That Are Insensitive to Catabolite Repression of Proline Degradation

In Salmonella typhimurium the two enzymes of proline catabolism, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase, are subject to catabolite repression when the cells are grown in the presence of glucose. Mutants partially relieved of catabolite repression (PutR) for the proline catabolic enzymes have been isolated by selection on agar plates containing glucose and proline. The specificity of the catabolite repression-insensitive character for the enzymes of proline utilization has been confirmed by an analysis of other unrelated catabolic enzymes. Histidase and amylomaltase of the mutant strains are equally as sensitive to glucose repression as are the enzymes from the wild type. All four PutR mutants exhibit higher induced and higher basal levels of proline oxidase as compared with the corresponding wild-type levels. The mutations of three strains tested are cotransducible with constitutive, pleiotrophic-negative and structural gene mutations of the put region. Three-factor crosses indicate that two putR mutations are located at one end of the cluster of put mutations.     

Vibrio cholerae Cytolysin Causes an Inflammatory Response in Human Intestinal Epithelial Cells That Is Modulated by the PrtV Protease

Background

Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV.

Methodology/Principal Findings

Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-α (TNF-α) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis.

Conclusions/Significance

We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-α in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.     

Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.     

A cross-continental test of the Enemy Release Hypothesis: leaf herbivory on Acer platanoides (L.) is three times lower in North America than in its native Europe

Acer platanoides (Norway maple) is a widespread native tree species in Europe. It has been introduced to North America where it has often established dense stands in both secondary woodlands and relatively undisturbed mature woodlands. In Europe A. platanoides is also extending its original range, but generally seems to exist at much lower densities. One explanation for the ‘aggressiveness’ of invasive plants such as A. platanoides is that they have left behind pests and diseases which limit their population densities in their native lands (the enemy release hypothesis or ERH). To assess the ERH for Norway maple, a large network of collaborators assessed leaf herbivory rates in populations throughout Europe and North America. We found significantly lower total leaf herbivory (1.6% ± 0.19, n = 21 vs. 7.4% ± 1.94, n = 34) and lower fungal damage (1.0% ± 0.35, n = 13 vs. 3.7% ± 0.85, n = 34) in North America than in Europe over a 2 year period, which is consistent with the predictions of the Enemy Release Hypothesis. Across years, the average total leaf herbivory was significantly correlated with average annual temperature of the site (P < 0.05), although this was mostly due to sites in Europe (P < 0.001), and not sites in North America (P > 0.05). Furthermore, only populations in Europe showed very high levels of herbivory (e.g., nine sites had total leaf herbivory ranging from 10.0 to 51.2% in at least 1 year) or leaf fungal damage (only one site in North America showed high levels of fungal damage in 1 year), suggesting the possibility of more frequent episodic outbreaks in the native range. Leaf herbivory and fungal damage are only two aspects of consumer pressure and we do not know whether the differences reported here are enough to actually elicit release from top-down population control, but such large scale biogeographic differences in herbivory contribute towards understanding exotic invasions. Jonathan M. Adams and Wei Fang—equally contributed as first authors. A list of the participating members of the Transatlantic Acer platanoides Invasion Network is given in the Appendix 3.     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.