Global diversity of cumaceans &; tanaidaceans (Crustacea: Cumacea &; Tanaidacea) in freshwater

Cumacea and Tanaidacea are marginal groups in continental waters. Although many euryhaline species from both groups are found in estuaries and coastal lagoons, most occur only temporarily in non-marine habitats, appearing unable to form stable populations there. A total of 21 genuinely non-marine cumaceans are known, mostly concentrated in the Ponto-Caspian region, and only four tanaids have been reported from non-marine environments. Most non-marine cumaceans (19 species) belong in the Pseudocumatidae and appear restricted to the Caspian Sea (with salinity up to 13‰) and its peripheral fluvial basins, including the northern, lower salinity zones of the Black Sea (Sea of Azov). There are nine Ponto-Caspian genera, all endemic to the region. Only two other taxa (in the family Nannastacidae) occur in areas free of any marine–water influence, in river basins in North and South America. Both seem able to survive in waters of raised salinity of the lower reaches of these fluvial systems; but neither has been recorded in full salinity marine environments. The only non-marine tanaidacean thus far known lives in a slightly brackish inland spring in Northern Australia. The genus includes a second species, from a brackish-water lake at the Bismarck Archipelago, tentatively included here as non-marine also. Two additional species of tanaidaceans have been reported from non-marine habitats but both also occur in the sea. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

Clinanthus microstephium,an Amaryllidaceae Species with Cholinesterase Inhibitor Alkaloids: Structure−Activity Analysis of Haemanthamine Skeleton Derivatives

Plants of the Amaryllidaceae family are well‐known (not only) for their ornamental value but also for the alkaloids that they produce. In this report, the first phytochemical study of Clinanthus genus was carried out. The chemical composition of alkaloid fractions from Clinanthus microstephium was analyzed by GC/MS and NMR. Seven known compounds belonging to three structural types of Amaryllidaceae alkaloids were identified. An epimeric mixture of a haemanthamine‐type compound (6‐hydroxymaritidine) was tested as an inhibitor against acetyl‐ and butyrylcholinesterase enzymes (AChE and BChE, respectively), two enzymes relevant in the treatment of Alzheimer's disease, with good results. Structure–activity relationships through molecular docking studies with this alkaloid and other structurally related compounds were discussed.     

The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

Arbuscular Mycorrhizal Symbiosis with Arundo donax Decreases Root Respiration and Increases Both Photosynthesis and Plant Biomass Accumulation

The effect of arbuscular mycorrhiza (AM) symbiosis on plant growth is associated with the balance between costs and benefits. A feedback regulation loop has been described in which the higher carbohydrate cost to plants for AM symbiosis is compensated by increases in their photosynthetic rates. Nevertheless, plant carbon balance depends both on photosynthetic carbon uptake and respiratory carbon consumption. The hypothesis behind this research was that the role of respiration in plant growth under AM symbiosis may be as important as that of photosynthesis. This hypothesis was tested in Arundo donax L. plantlets inoculated with Rhizophagus irregularis and Funneliformis mosseae. We tested the effects of AM inoculation on both photosynthetic capacity and in vivo leaf and root respiration. Additionally, analyses of the primary metabolism and ion content were performed in both leaves and roots. AM inoculation increased photosynthesis through increased CO₂ diffusion and electron transport in the chloroplast. Moreover, respiration decreased only in AM roots via the cytochrome oxidase pathway (COP) as measured by the oxygen isotope technique. This decline in the COP can be related to the reduced respiratory metabolism and substrates (sugars and tricarboxylic acid cycle intermediates) observed in roots.     

The b-32 protein from maize endosperm: characterization of genomic sequences encoding two alternative central domains

As derived from a cDNA clone, the structure of the b-32 protein ofZea mays, a putative regulatory factor of zein expression, has a central acidic region separated by two domains covered by secondary structure motifs. In this work, three b-32 genomic clones were selected from two genomic libraries obtained from the maize inbred lines W64A and A69Y. The nucleotide sequences of the complete coding region of eachb-32 gene, as well as long stretches of their 5 and 3 flanking regions, were determined. Introns are not present in the b-32 genomic sequences. Minor variations among the three genes and an earlier reported b-32 cDNA indicates that they constitute a gene family showing a characteristic polymorphism. Such a polymorphism is highly evident in large segments of the upstream regulatory sequences. Interestingly, when compared with cDNA (W64A) or with geneb-32.120 (W64A), the genesb-32.129 (W64A) andb-32.152 (A69Y) show three jumps of the reading frame in the central part of the coding region, resulting in a completely different sequence of the b-32 protein central domain. In all cases, variations in the N- and C-terminal domains account only for microheterogeneity.     

Regulation of K+ and NO3− fluxes in roots of sunflower (Helianthus annuus) after changes in light intensity

Shoot activity has been reported to affect rates of ion uptake by plant roots in other ways than merely through supply of assimilates. To elucidate the mechanisms by which a signal from the upper part of the plant controls the rate of K⁺ and NO₃⁻ uptake by roots, both uptake of K⁺ and NO₃⁻ and secretion into the xylem of young sunflower plants ( Helianthus annuus L.) were measured after changes in light intensity.
No close correlation was observed between the uptake of NO₃⁻ and that of K⁺; an increase in light intensity produced a much greater stimulation of NO₃⁻ uptake than of K⁺ uptake. On the other hand, secretion of NO₃⁻ into the xylem was tightly coupled to that of K⁺, and this coupling was strongly disturbed by excision of the root. The results suggest the involvement of the K₂-malate shuttle on the regulation by the shoot of K⁺ and NO₃⁻ secretion in the xylem, which is linked to NO₃⁻ uptake, while K⁺ uptake is independent of this regulation mechanism.     

Planarian pharynx regeneration in regenerating tail fragments monitored with cell-specific monoclonal antibodies

 The special morphological features of freshwater planarians make them an attractive and informative model for studying the processes of regeneration and pattern formation. In this work, we investigate pattern formation and maturation of the planarian pharynx during regeneration in tail fragments. Using three monoclonal antibodies (TCAV-1, TF-26 and TMUS-13) specific for epithelial, secretory and muscle cells, respectively, we followed the sequence and timing of differentiation and maturation of these three cell types within the regenerating pharynx. Two of these monoclonal antibodies, TCAV-1 and TMUS-13, also labelled morphologically immature cells that appear to be committed to the differentiation pathway leading to their respective adult cell types. Our results show that the cells forming the new pharynx come from undifferentiated cells through proliferation and differentiation processes rather than from differentiated cells of the old stump. We describe three stages of pharynx regeneration according to the immunoreactivity shown: (1) no immunoreactivity, corresponding to the accumulation of undifferentiated cells that form the pharynx primordium; (2) immunoreactivity to TCAV-1 and TMUS-13, corresponding to the re-building of the pharynx; and (3) immunoreactivity to TF-26, corresponding to a fully mature and functional pharynx. The sequence of differentiation of these three cell types suggests that the pharynx grows by intercalation of new undifferentiated cells coming from the parenchyma between the older pharyngeal cells, in agreement with existing models of pharynx regeneration. Finally, our results suggest an intercalary model for pharynx epithelial cell renewal. Received: 30 September 1996 / Accepted: 6 December 1996     

Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody drug conjugation

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro‐domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine‐scan of the mTGase pro‐domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro‐domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro‐domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30–75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site‐specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.     

A minimized motile machinery for Mycoplasma genitalium

The cell wall‐less bacterium Mycoplasma genitalium uses specialized adhesins located at the terminal organelle to adhere to host cells and surfaces. The terminal organelle is a polar structure protruding from the cell body that is internally supported by a cytoskeleton and also has an important role in cell motility. We have engineered a M. genitalium null mutant for MG491 protein showing a massive downstream destabilization of proteins involved in the terminal organelle organization. This mutant strain exhibited striking similarities with the previously isolated MG_218 null mutant strain. Upon introduction of an extra copy of MG_318 gene in both strains, the amount of main adhesins P140 and P110 dramatically increased. These strains were characterized by microcinematography, epifluorescence microscopy and cryo‐electron microcopy, revealing the presence of motile cells and filaments in the absence of many proteins considered essential for cell adhesion and motility. These results indicate that adhesin complexes play a major role in the motile machinery of M. genitalium and demonstrate that the rod element of the cytoskeleton core is not the molecular motor propelling mycoplasma cells. These strains containing a minimized motile machinery also provide a valuable cell model to investigate the adhesion and gliding properties of this human pathogen.