Hydration and selfassociation of haemoglobin in solution

The hydration of haemoglobin was determined by the nmr-freezing technique. There is a hydration-threshold around 15 mM(haem)/L, with the maximum hydration of ～ 0.46 g H₂O/g Hb. The phosphate buffered solutions show a completely different behaviour from the other ionic and deionized solutions. The results in ordinary water and in the presence of 80% ²H₂O are indistin-guishable. The hydration is neither a function of the extremes in the ionic double layer structure nor of the type of ions. The threshold Hb-concentration does not depend on the absolute amount of hydration and the type of ion. The results corroborate the selfassociation of haemoglobin in concentrated solutions.     

Mycobacteria in water and loose deposits of drinking water distribution systems in Finland

Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 x 10(5) and 3.9 x 10(5) CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to Mycobacterium lentiflavum, M. tusciae, M. gordonae, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.     

The Yellow Gorgonian Eunicella cavolini: Demography and Disturbance Levels across the Mediterranean Sea

The yellow octocoral Eunicella cavolini is one of the most common gorgonians thriving in Mediterranean hard-bottom communities. However, information regarding its distribution and ecology in several parts of the Mediterranean is lacking, while population trends and conservation status remain largely unknown. We investigated 19 populations of E. cavolini over three representative geographic regions: the NW Mediterranean, CE Adriatic, and N Aegean. Focusing on the upper bathymetric range of the species (<40 m), data were collected on the populations’ upper depth limit, density, colony height, and extent of injury. A three-level hierarchical sampling design was applied to assess the existence of spatial patterns, using: a) regions (located thousands of km apart), b) localities within regions (tens to hundreds of km apart), and c) sites within localities (hundreds of m to a few km apart). In the NW Mediterranean and CE Adriatic, the upper distribution limit was at depths ≤15 m, whereas in the N Aegean most populations were found deeper than 30 m. Population density ranged between 4.46-62 colonies per m², while mean colony height was 15.6±8.9 SD cm with a maximum of 62 cm. The NW Mediterranean sites were characterized by dense populations dominated by small colonies (<20 cm), periodic recruitment, and low proportion of large gorgonians (>30 cm). The CE Adriatic displayed intermediate densities, with well-structured populations, and continuous recruitment. In the N Aegean, most populations presented low densities, high proportion of large colonies, but low number of small colonies, signifying limited recruitment. Disturbance levels, as a function of extent and type of injury, are discussed in relation to past or present human-induced threats. This work represents geographically the most wide ranging demographic study of a Mediterranean octocoral to date. The quantitative information obtained provides a basis for future monitoring at a Mediterranean scale.     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

Ecoregion-Based Conservation Planning in the Mediterranean: Dealing with Large-Scale Heterogeneity

Spatial priorities for the conservation of three key Mediterranean habitats, i.e. seagrass Posidonia oceanica meadows, coralligenous formations, and marine caves, were determined through a systematic planning approach. Available information on the distribution of these habitats across the entire Mediterranean Sea was compiled to produce basin-scale distribution maps. Conservation targets for each habitat type were set according to European Union guidelines. Surrogates were used to estimate the spatial variation of opportunity cost for commercial, non-commercial fishing, and aquaculture. Marxan conservation planning software was used to evaluate the comparative utility of two planning scenarios: (a) a whole-basin scenario, referring to selection of priority areas across the whole Mediterranean Sea, and (b) an ecoregional scenario, in which priority areas were selected within eight predefined ecoregions. Although both scenarios required approximately the same total area to be protected in order to achieve conservation targets, the opportunity cost differed between them. The whole-basin scenario yielded a lower opportunity cost, but the Alboran Sea ecoregion was not represented and priority areas were predominantly located in the Ionian, Aegean, and Adriatic Seas. In comparison, the ecoregional scenario resulted in a higher representation of ecoregions and a more even distribution of priority areas, albeit with a higher opportunity cost. We suggest that planning at the ecoregional level ensures better representativeness of the selected conservation features and adequate protection of species, functional, and genetic diversity across the basin. While there are several initiatives that identify priority areas in the Mediterranean Sea, our approach is novel as it combines three issues: (a) it is based on the distribution of habitats and not species, which was rarely the case in previous efforts, (b) it considers spatial variability of cost throughout this socioeconomically heterogeneous basin, and (c) it adopts ecoregions as the most appropriate level for large-scale planning.     

The growth pattern of transplanted normal and nodular hepatocytes

Overt neoplasia is often the end result of a long biological process beginning with the appearance of focal lesions of altered tissue morphology. While the putative clonal nature of focal lesions has often been emphasized, increasing attention is being devoted to the possible role of an altered growth pattern in the evolution of carcinogenesis. Here we compare the growth patterns of normal and nodular hepatocytes in a transplantation system that allows their selective clonal proliferation in vivo. Rats were pre-treated with retrorsine, which blocks the growth of resident hepatocytes, and were then transplanted with hepatocytes isolated from either normal liver or hepatocyte nodules. Both cell types were able to proliferate extensively in the recipient liver, as expected. However, their growth pattern was remarkably different. Clusters of normal hepatocytes integrated in the host liver, displaying a normal histology; however, transplanted nodular hepatocytes formed new hepatocyte nodules, with altered morphology and sharp demarcation from surrounding host liver. Both the expression and distribution of proteins involved in cell polarity, cell communication, and cell adhesion, including connexin 32, E-cadherin, and matrix metalloproteinase-2, were altered in clusters of nodular hepatocytes. Furthermore, we were able to show that down-regulation of connexin 32 and E-cadherin in nodular hepatocyte clusters was independent of growth rate. These results support the concept that a dominant pathway towards neoplastic disease in several organs involves defect(s) in tissue pattern formation.     

Lipopolysaccharide-induced anti-inflammatory acute phase response is enhanced in spermidine/spermine N 1-acetyltransferase (SSAT) overexpressing mice

Bacterial lipopolysaccharide (LPS) is an effective activator of the components of innate immunity. It has been shown that polyamines and their metabolic enzymes affect the LPS-induced immune response by modulating both pro- and anti-inflammatory actions. On the other hand, LPS causes changes in cellular polyamine metabolism. In this study, the LPS-induced inflammatory response in spermidine/spermine N(1)-acetyltransferase overexpressing transgenic mice (SSAT mice) was analyzed. In liver and kidneys, LPS enhanced the activity of the polyamine biosynthetic enzyme ornithine decarboxylase and increased the intracellular putrescine content in both SSAT overexpressing and wild-type mice. In survival studies, the enhanced polyamine catabolism and concomitantly altered cellular polyamine pools in SSAT mice did not affect the LPS-induced mortality of these animals. However, in the acute phase of LPS-induced inflammatory response, the serum levels of proinflammatory cytokines interleukin-1β and interferon-γ were significantly reduced and, on the contrary, anti-inflammatory cytokine interleukin-10 was significantly increased in the sera of SSAT mice compared with the wild-type animals. In addition, hepatic acute-phase proteins C-reactive protein, haptoglobin and α(1)-acid glycoprotein were expressed in higher amounts in SSAT mice than in the wild-type animals. In summary, the study suggests that SSAT overexpression obtained in SSAT mice enhances the anti-inflammatory actions in the acute phase of LPS-induced immune response.     

Direct comparison of the Ames microplate format (MPF) test in liquid medium with the standard Ames pre-incubation assay on agar plates by use of equivocal to weakly positive test compounds

The Ames microplate format (MPF?) test, which uses liquid media and in 384-well microplates with a readout based on a colour-change, has been used for over 10 years at several major pharmaceutical companies for screening the genotoxic potential of early drug candidates when compound supply is minimal. Meanwhile, Xenometrix has adapted this screen from the two-strain Ames II test for use with five tester strains, in compliance with OECD Guideline 471. A set of 15 equivocal to weakly positive chemicals selected from the National Toxicology Program (NTP) database was tested simultaneously in the Ames microplate format (MPF) and the standard Ames pre-incubation method on agar plates. Such a direct comparison of the two test methods with the same overnight culture(s), chemicals and S9-mix preparation should exclude external variability factors. Thirteen of the 15 chemicals showed concordant results in both tests despite the choice of chemicals that showed varying inter- and even intra-laboratory results in the NTP studies. These results indicate that the Ames MPF? assay is a reliable predictive tool that can be used like the regular Ames test to evaluate compounds for mutagenicity.