Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

Localization of corticotropin-releasing factor-containing neurons in the brain of the domestic fowl

Summary The corticotropin-releasing factor (CRF)-containing neurons were investigated in the brain of the domestic fowl by means of the peroxidase-antiperoxidase technique at the light-microscopic level. The detection of CRF-immunoreactivity was facilitated by silver intensification. CRF-containing perikarya were found in the paraventricular, preoptic and mammillary nuclei of the hypothalamus and in some extrahypothalamic areas (nuclei dorsomedialis and dorsolateralis thalami, nucleus accumbens septi, lobus parolfactorius, periaqueductal gray of the mesencephalon, nucleus oculomotorius ventralis). Immunoreactive nerve fibers and terminals were demonstrated in the external zone of the median eminence and the organum vasculosum of the lamina terminalis. These results indicate that an immunologically demonstrable CRF-neurosecretory system also exists in the avian central nervous system.     

The fine structure of distal receptors on the labium of the aphid,Brevicoryne brassicae L. (Homoptera)

Summary Short peg receptors located at the distal tip of the aphid labium have the structure of mechanoreceptors. Each peg is innervated by a single sensory nerve which is anchored eccentrically to a basal cuticular tube and terminates in electron-dense material in the base of the peg. The arrangement and eccentric insertion of the eight pegs in the labial wall on one side of the stylet groove, with the eccentric insertions of their innervating neurones, provide a mirror image of the receptors on the opposite side. On the basis of a comparison of the structure of these receptors with that of tactile receptors for which electrophysiological data on sensitivity are available, it is possible to predict that the receptors detect both surface contact (pressure) and surface profile; and that the bilateral symmetry in the receptor arrangement facilitates the detection of vein contours which are preferred settling sites on the leaf. The structure of the dendritic terminal and its insertion is that of a well reinforced cytoskeleton designed to transmit tension to the cell membrane, in agreement with the concept that transduction is a membrane related phenomenon. The distal microtubules, fifty per-cent of which originate as well as terminate in the tubular body, are packed in electron-dense material which binds to the cell membrane. The membrane in turn is attached to cuticular components of the receptor. Abrupt changes in dimension of the dendritic outer segment may be designed to modulate the conduction of a membrane potential. On the other hand, lack of continuity in the microtubules makes these organelles poor candidates for the transduction of excitation from a distal site of stimulation to a proximal region.Supported by operating grants Nos. A 6063 and A 9856 from NRCC     

Characterization of the molecular properties of KtrC,a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K⁺) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K⁺ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.     

Aquaglyceroporins: Generalized metalloid channels

Background

Aquaporins (AQPs), members of a superfamily of transmembrane channel proteins, are ubiquitous in all domains of life. They fall into a number of branches that can be functionally categorized into two major sub-groups: i) orthodox aquaporins, which are water-specific channels, and ii) aquaglyceroporins, which allow the transport of water, non-polar solutes, such as urea or glycerol, the reactive oxygen species hydrogen peroxide, and gases such as ammonia, carbon dioxide and nitric oxide and, as described in this review, metalloids.

Scope of review

This review summarizes the key findings that AQP channels conduct bidirectional movement of metalloids into and out of cells.

Major conclusions

As(OH)₃ and Sb(OH)₃ behave as inorganic molecular mimics of glycerol, a property that allows their passage through AQP channels. Plant AQPs also allow the passage of boron and silicon as their hydroxyacids, boric acid (B(OH)₃) and orthosilicic acid (Si(OH)₄), respectively. Genetic analysis suggests that germanic acid (GeO₂) is also a substrate. While As(III), Sb(III) and Ge(IV) are toxic metalloids, borate (B(III)) and silicate (Si(IV)) are essential elements in higher plants.

General significance

The uptake of environmental metalloids by aquaporins provides an understanding of (i) how toxic elements such as arsenic enter the food chain; (ii) the delivery of arsenic and antimony containing drugs in the treatment of certain forms of leukemia and chemotherapy of diseases caused by pathogenic protozoa; and (iii) the possibility that food plants such as rice could be made safer by genetically modifying them to exclude arsenic while still accumulating boron and silicon. This article is part of a Special Issue entitled Aquaporins.