Culture on sugar medium enhances photosynthetic capacity and high light resistance of plantlets grown in vitro

The significance of photosynthetic photon flux (PPF) and sugar feeding for the production of plants in vitro is only poorly understood. Nicotiana tabacum L. plantlets were grown photoautotrophically and photomixotrophically (3% sucrose) at two different PPFs (60 µmol m⁻² s⁻¹ and 200 µmol m⁻² s⁻¹) to investigate the effect of these culture parameters on photosynthetic performance and growth. Photomixotrophically‐grown plantlets showed an increase in carbohydrate content, mainly in glucose and fructose. Plant growth, dry matter accumulation and total leaf area were higher under photomixotrophic than photoautotrophic conditions. Not only biomass formation but also photosynthesis was positively affected by exogenous sucrose; the chlorophyll (Chl) content and the light‐saturated rate of photosynthetic oxygen evolution were higher in photomixotrophic plantlets. Photoinhibition occurred in plantlets that were grown photoautotrophically at the higher PPF. It became apparent as a loss in Chl content and photochemical efficiency. Photoinhibited plantlets showed a decrease in the D2/LHCII and CP47/LHCII ratios, suggesting a preferential loss of proteins from the photosystem II (PSII) core. The increased content of xanthophyll cycle pigments in photoinhibited plantlets indicated that also protective mechanisms were activated. Photomixotrophic growth of the plantlets prevented the occurrence of photoinhibitory symptoms. Therefore, we conclude that culture on sugar medium increases not only the photosynthetic potential but also the high light resistance of plantlets grown in vitro.     

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.     

A naturally occurring highly attenuated strain of type 1 poliomyelitis virus

Summary Twenty strains of poliomyelitis virus recovered from cases of abortive, non-paralytic, poliomyelitis have been tested for neurotropism. Only one strain possessed a low intraspinal activity inCynomolgus monkeys. Six single particle cultures were derived from this naturally occurring highly attenuated strain of type 1 poliomyelitis virus. It has been shown that the original virus consisted of a mixture of virus particles with varying degree of neurotropism. The antigenic and immunizing capacity of one highly attenuated plaque progeny has been tested in guinea pigs and monkeys. Aided with a grant from the National Health Research Council T.N.O.     

Microwave heating as an energy source for the predrying of herbage samples

Summary Preliminary experiments have shown that a commercial microwave cooking range can give a very rapid low-temperature drying of herbage samples. Its throughput can compete with hot-air ovens used for laboratory drying. The capital cost of the equipment will resrict its use to samples for chemical or biochemical analysis. The samples obtained are similar to freeze-dried samples.     

Invasive round goby Neogobius melanostomus has sex‐dependent locomotor activity and is under‐represented in catches from passive fishing gear compared with seine catches

The higher proportion of males of the invasive round goby Neogobius melanostomus in samples from two activity selective passive fishing gears compared with one activity non‐selective fishing gear in three Dutch lakes is related to higher male locomotory activity and is a sex‐dependent trait. This difference in activity reflects the different ecology of male and female N. melanostomus.     

Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.     

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe²²⁹ and Phe²⁷⁹ of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe²⁷⁹. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe²⁷⁹. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe²⁷⁹, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe²²⁹.     

Timber stacks: potential ecological traps for an endangered saproxylic beetle,the Rosalia longicorn <Emphasis Type="Italic">Rosalia alpina</Emphasis>

Ecological traps are serious, anthropogenic threats to animal populations. However, in certain cases it is difficult to determine whether they really act in the expected manner. This applies to the harmful effects of beech timber stacked in forests on the endangered saproxylic beetle Rosalia longicorn Rosalia alpina, which have been mentioned in numerous scientific articles, conservation action plans and similar publications. The aim of this paper is to determine whether beech timber stacks meet the criteria of an ecological trap for the Rosalia longicorn. Two basic criteria of such a trap are analysed: the attractiveness of timber stacks and the impossibility of complete larval development. The results show that beech timber stacks are highly attractive to Rosalia longicorn imagines. Moreover, the time during which the timber is stacked is shown to be significantly shorter than the species’ larval development period. These results suggest that timber stacks can be treated as operative ecological traps for the Rosalia longicorn, even though the extent of their influence on the demographic parameters of this beetle’s population has not been estimated. Forest management practices, i.e. increasing amounts and shifts in timing of wood storage, could intensify this threat.     

Expression and activity of cGMP-dependent phosphodiesterases is up-regulated by lipopolysaccharide (LPS) in rat peritoneal macrophages

It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.     

Lack of internucleosomal DNA fragmentation is related to Cl(-) efflux impairment in hematopoietic cell apoptosis.

The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase-dependent release of ICAD/DFF-45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperosmotic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50-150 kb) DNA cleavage is abolished if Cl(-) efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl(-) channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti-Fas antibody). In these conditions, all the apoptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF-45. In vitro assays with lysates of cells in which Cl(-) efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF-45 processing per se is sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl(-) efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.