Peanut (Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

Metal resistance in Acinetobacter and its relation to β-lactamase production

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce -lactamase. Fifty two percent of the strains produced -lactamase. -Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in -lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced -lactamase. Sensitivity and resistance to copper and cadium was equally distributed between -lactamase producers and non-producers. -Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.     

Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

Summary Post-mitotic epidermal cells of barley leaves were found to contain, in addition to cortical microtubules (CMTs), distinct arrays of endoplasmic microtubules (EMTs). These encircle nuclei and continuously merge into the CMT arrays that underly the plasmalemma. Detailed three-dimensional reconstruction of both types of MTs during fungal infection showed that profound and very rapid MT rearrangements occurred especially in the case of incompatible (resistant) barley-powdery mildew genotype combination. The most early MT responses, followed by their subsequent complete disintegration, were recorded around nuclei. These events might be relevant for the induction of such nuclear processes as onset of DNA synthesis and nuclear chromatin condensation. Observed pattern of early infection events, as well as less prominent responses in the case of compatible (susceptible) barley-powdery mildew genotype combination, both findings suggest that rapid reorganization of the MT cytoskeleton could be involved in recognition of the fungus by host cells and in the initiation of resistance responses in barley leaves. We hypothesize that the integrity and dynamics of the MT cytoskeleton, especially of its perinuclear part, might participate in control mechanisms involved in activation of resistance genes.Abbreviations CMTs cortical microtubules - EMTs endoplasmic microtubules - MT microtubules - PI propidium iodide - SC sensitive combination - RC resistant combination     

Eukaryote-conserved histone post-translational modification landscape in Giardia duodenalis revealed by mass spectrometry

Diarrheal disease caused by Giardia duodenalis is highly prevalent, causing over 200 million cases globally each year. The processes that drive parasite virulence, host immune evasion and transmission involve coordinated gene expression and have been linked to epigenetic regulation. Epigenetic regulatory systems are eukaryote-conserved, including in deep branching excavates such as Giardia, with several studies already implicating histone post-translational modifications in regulation of its pathogenesis and life cycle. However, further insights into Giardia chromatin dynamics have been hindered by a lack of site-specific knowledge of histone modifications. Using mass spectrometry, we have provided the first known molecular map of histone methylation, acetylation and phosphorylation modifications in Giardia core histones. We have identified over 50 previously unreported histone modifications including sites with established roles in epigenetic regulation, and co-occurring modifications indicative of post-translational modification crosstalk. These demonstrate conserved histone modifications in Giardia which are equivalent to many other eukaryotes, and suggest that similar epigenetic mechanisms are in place in this parasite. Further, we used sequence, domain and structural homology to annotate putative histone enzyme networks in Giardia, highlighting representative chromatin modifiers which appear sufficient for identified sites, particularly those from H3 and H4 variants. This study is to our knowledge the first and most comprehensive, complete and accurate view of Giardia histone post-translational modifications to date, and a substantial step towards understanding their associations in parasite development and virulence.     

Characterization of archaeal symbionts of sponges from the coral reef ecosystems of the Gulf of Mannar,Southeast coast of India

Sponges accommodate a diverse group of microorganisms with varied metabolic capabilities. The bacterial associates of sponges are widely studied while our understanding of archaeal counterparts is scanty. In the present study, we report the archaeal associates of two sponges, Pseudoceratina purpurea (NCBI barcode: KX454492) and Cinachyra sp. (NCBI barcode: KX454495), found in the coral reef ecosystems of Gulf of Mannar, India. Archaea in the water column was predominated by members of class Halobacteria of Phylum Euryarchaeota (97%) followed by a minor fraction (3%) of Nitrosopumilus sp. of phylum Thaumarchaeota. Interestingly, Thaumarchaeota was identified as the sole archaeal population associated with the two sponges studied, among which Nitrosopumilus sp. occuppied 80 and 100% of the sequences in the clone library of P. purpurea and Cinachyra sp. respectively. Other archaea found in the P. purpurea were Nitrososphaera (10%) and unclassified ones (10%). The study identified Nitrosopumilus sp. as a unique symbiotic archaeon of sponges, P. purpurea and Cinachyra sp. The existence of host driven factors in selecting specific associates from a diverse group of archaea in the environment may need further investigations.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

Mutational analysis of TYR gene and its structural consequences in OCA1A

Oculocutaneous albinism type 1A (OCA1A) is the most severe form of albinism characterized by a complete lack of melanin production throughout life and is caused by mutations in the TYR gene. TYR gene codes tyrosinase protein to its relation with melanin formation by knowing the function of these SNPs. Based on the computational approaches, we have analyzed the genetic variations that could change the functional behaviour by altering the structural arrangement in TYR protein which is responsible for OCA1A. Consequences of mutation on TYR structure were observed by analyzing the flexibility behaviour of native and mutant tyrosinase protein. Mutations T373K, N371Y, M370T and P313R were suggested as high deleterious effect on TYR protein and it is responsible for OCA1A which were also endorsed with previous in vivo experimental studies. Based on the quantitative assessment and flexibility analysis of OCA1A variants, T373K showed the most deleterious effect. Our analysis determines that certain mutations can affect the dynamic properties of protein and can lead to disease conditions. This study provides a significant insight into the underlying molecular mechanism involved in albinism associated with OCA1A.     

Plant Neurobiology as a Paradigm Shift Not Only in the Plant Sciences

Plants are complex living beings, extremely sensitive to environmental factors, continuously adapting to the ever changing environment. Emerging research document that plants sense, memorize, and process experiences and use this information for their adaptive behavior and evolution. As any other living and evolving systems, plants act as knowledge accumulating systems. Neuronal informational systems are behind this concept of organisms as knowledge accumulating systems because they allow the most rapid and efficient adaptive responses to changes in environment. Therefore, it should not be surprising that neuronal computation is not limited to animal brains but is used also by bacteria and plants. The journal, Plant Signaling & Behavior, was launched as a platform for exchanging information and fostering research on plant neurobiology in order to allow our understanding of plants in their whole integrated, communicative, and behavioral complexity.

I always go by official statistics because they are very carefully compounded and, even if they are false, we have no others …∼ Jaroslav Hašek, 1911

Key Words: plant neurobiology, sensory biology, behavior, biological complexity, evolution, signal integrationThis quotation of writer and mystificator Jaroslav Hašek is from his electorial speech aimed to get a seat in the Austro-Hungarian parliament for his imaginary political party ‘Moderate Progress within the Limits of the Law’ in 1911. It indicates how statistics can be misused for manipulation of public opinion, sometimes allegedly for general good. This quotation is partially relevant also for recent biology which is passing through a critical cross-road from reductionist-mechanistic concepts and methodologies towards the post-genomic, holistic, systems-based analysis of integrated and communicative hierarchic networks known as life processes.There is a message hidden in this Hašek''s aphorism. All those mathematical models, scientific theories and concepts, however appealing, harmonious and long-standing … but which do not correspond to reality …; inevitably will be ‘killed by ugly’ facts generated by scientific progress, and finally replaced by new models, theories, and concepts.¹Despite the indisputable success of the reductionistic approach in providing many discoveries regarding single cells and their components, it is increasingly clear that promises of ‘mechanistic’ genocentric biology were just chimeras and that living organisms are much more complex than the sum of their constituents. Ernst Mayr, in his final opus, almost a testament published at his age of 100, strongly opposed the belief that the reductionism at the molecular level could help to explain the complexity of life. He stressed that the concept of biological “emergence”, which deals with the occurrence of unexpected features in complex living systems, is not fully accessible using only physical and chemical approaches.²Themes of hierarchy, continuity, and order dominated biology before the turn of the century, but these have in many cases been replaced by images of the workshop. Examples include such terms as ‘machineries’, ‘mechanistic understanding’, ‘mechanistic explanation’, ‘motors’, ‘machines’, ‘clocks’ etc. This shift may well reflect the characteristic style of our age. These concepts, although useful for mining of details, do not reveal the true complexity of life and can be misleading. Even a one-celled organism is made up of ‘millions’ of subcellular parts. Concerning the great complexity of unicellular creatures Ilya Prigogine (1973) wrote: “… but let us have no illusions, our research would still leave us quite unable to grasp the extreme complexity of the simplest of organism.”³ Moreover, eukaryotic cell proved to be, in fact, ‘cells within cell’,^4–8 while there are numerous supracellular situations, the most dramatic one is represented by plants when all cells are interconnected via plasmodesmata into supracellular organism.⁶ All this collectively indicate that the currently valid ‘Cell Theory’ dogma is approaching its replacement with a new updated concept of a basic unit of eukaryotic life.^6–8All those mathematical models, scientific theories and concepts, however appealing, harmonious and long-standing … but which do not correspond to reality …; inevitably will be ‘killed by ugly’ facts generated by scientific progress, and finally replaced by new models, theories, and concepts.Furthermore, genomes are much more complex and dynamic as we ever anticipated.^9,10 They often have as much as 99% of non-coding DNA sequences,¹¹ which is not ‘junk DNA’ but rather DNA which is part of multitask networks integrating coding DNA.¹² In genomes exposed to stress (like mutations), changes are scored preferentially in non-coding sequences which regain a new balance by complex changes in genome composition and activity.^9,10,13,14 There are several definitions regarding what is gene¹¹ and molecular biologists and genetics are learning to be careful not to make strong conclusions from under-expression, knocking-out, or overexpression of any particular gene. It is increasingly clear that mutations in single genes are accompanied with altered expressions of other genes and non-coding DNA sequences too, and even subtle re-arrangements of chromatin structure and genome architecture are possible. The dynamic genome actively regains the lost balance, also via extensive re-shufflings of non-coding DNA.After complete sequencing of numerous genomes, it is clear that our understanding of what constitutes life and what distinguishes living biological systems from non-living chemical - biochemical systems is not much better than our understanding before the start of the genomics era some 60 years ago. Yet, it is also obvious that living systems, whether single cells or whole complex organisms like animals and plants, are not machines and automata which respond to external signals via a limited set of predefined responses and automatic reflexes. While humans and other animals, even insects, are already out of this ‘mechanistic’ trap^15,16 which can be traced back to Descartes,¹⁷ plants are still considered to act only in predetermined automatic fashions, as mechanical devices devoid of any possibility for choice and planning of their activities. In contrast to machines, life systems are based on wet chemistry, being systems of hierarchical and dynamic integration, communication and emergence.^1,18Recently, a critical mass of data has accumulated demanding reconsideration of this mechanistic view of plants.^19,20 Plants are complex living beings, extremely sensitive to environmental factors and continuously adapting to the ever changing environment.²¹ In addition, plants respond to environmental stimuli as integrated organisms. Often, plants make important decisions, such as onset or breakage of dormancy and onset of flowering, which implicate some central or decentralized command center. Moreover, roots and shoots act in an integrated manner allowing dynamic balance of above-ground and below-ground organs. The journal, Plant Signaling & Behavior, was launched as a platform for exchange of information about the integration of discrete processes, including subcellular signalling integrated with higher-level processes. Signal integration and communication results in adaptive behavior of whole supracellular organisms, encompassing also complex, and still elusive, plant-plant, plant-insect, and plant-animal communications. Coordinated behavior based on sensory perception is inherent for neurobiological systems.²² Therefore, plants can be considered for neuronal individuals. Moreover, plants are also able to share knowledge perceived from environment with other plants, communicating both private and public messages.^23,24 This implicates social learning and behavioral inheritance in plants too.After complete sequencing of numerous genomes, it is clear that our understanding of what constitutes life and what distinguishes living biological systems from non-living chemical - biochemical systems is not much better than our understanding before the start of genomics era some 60 years ago.

Behavior

1. An activity of a defined organism: observable activity when measurable in terms of quantitative effects of the environment whether arising from internal or external stimuli.
2. Anything that an organism does that involves action and response to stimulation.

(Webster Third New International Dictionary 1961).Neuronal informational systems allow the most rapid and efficient adaptive responses. Therefore, it should not be surprising that neuronal computation is not limited to animal brains but is used also by bacteria and plants.Some of our colleagues assert that plants do not exhibit any integrated neuronal principles.²⁵ They maintain that plants do not show complex experience- or learning-based behavior. Plants, they aver, act rather as machines manifesting predefined reflexes. Yet recent studies indicate that even prokaryotic bacteria exhibit cognitive behavior^26,27 and posses linguistic communication and rudimentary intelligence.^28–30 Therefore, it should not be too surprising that plants also show features of communication and even plant-specific cognition.^{19,20,31,32–35} As any other living systems, plants act as ‘knowledge accumulating systems’.¹ In fact, in order to adapt, all organisms continuously generate hypotheses about their environment via well formulated ‘questions’ which are solved by an increasing set of possible ‘answers’ in order to adapt.¹ Neuronal informational systems are behind this concept of organisms as ‘knowledge accumulating systems’ because they allow the most rapid and efficient adaptive responses.²² As a consequence, neuronal computation is not limited to animal brains but is used also by bacteria and plants.Reductionistic approaches will continue to “atomize” biological systems. Nevertheless, the avalanche of new data will be in need of functional integration, winning adherents to the idea that plants have integrated signaling and communicative systems that endowed them with complex and adaptive behavior. We trust that Plant Signaling & Behavior, will become an important platform for exchange of these ideas. With progress of sciences, plants show more and more similarities to animals despite obviously plant-specific evolutionary origins, cellular basis, and multicellularity. We can just mention sexuality and sex organs, embryos, stem cells, immunity, circadian rhythms, hormonal and peptide signaling, sensory perception and bioelectricity including action potentials, communication and neurobiological aspects of signal integration. The whole picture strongly suggest that convergent evolution is much more important^36,37 than currently envisioned in evolutionary theories.Reductionistic approaches will continue to “atomize” biological systems. Nevertheless, the avalanche of new data will be in need of functional integration, winning adherents to the idea that plants have integrated signaling and communicative systems that endowed them with complex and adaptive behavior.We have started with Jaroslav Hašek and we close with him as well. His quotation from 1911 is also a warning for future that we should stay open-minded. We should not slip into dogmatic ‘traps’ which have been so characteristic for the mechanistic and genocentric biology. Mathematics and computational biology are important tools, and surely will play decisive role in systems biology in the future. But they can be easily misinterpreted, and even misused. Plant systems biology, and the whole biology in general, must overcome dogmas of mechanistic genocentric biology. We hope that characterizing plants in their whole behavioral and communicative complexity will allow us to better understand what is life and how it emerged from chemical and biochemical complex systems.     

Calcium dynamics during fertilization in C. elegans

Background

Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.

Results

Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed.

Conclusion

Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.