The Prohead-I structure of bacteriophage HK97: implications for scaffold-mediated control of particle assembly and maturation

Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in Escherichia coli, the delta domain guides 420 copies of the subunit into a procapsid with T = 7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it cannot mature further. When the virally encoded protease (gp4) is coexpressed with gp5, it is incorporated into the capsid and digests the delta domain followed by autoproteolysis to produce the metastable Prohead-II. Prohead-I^+P was isolated by coexpressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I^+P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I^+P was determined at 5.2 Å resolution, and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I^+P differed from Prohead-II due to the presence of the delta domain and the resulting repositioning of the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.     

Characterization of anti-ECA antibodies in rabbit antiserum against rough <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> O:3

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA_PG) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA_LPS); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA_PG and ECA_LPS. To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain “monovalent” anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECAPG, and this suggested that in Y. enterocolitica O:3 ECA_LPS only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA_PG and ECA_LPS expression were found to be regulated by temperature and repressed at 37°C.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFβ, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer

During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.     

Neural circuitry that governs Drosophila male courtship behavior

Male-specific fruitless (fru) products (Fru(M)) are both necessary and sufficient to "hardwire" the potential for male courtship behavior into the Drosophila nervous system. Fru(M) is expressed in approximately 2% of neurons in the male nervous system, but not in the female. We have targeted the insertion of GAL4 into the fru locus, allowing us to visualize and manipulate the Fru(M)-expressing neurons in the male as well as their counterparts in the female. We present evidence that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent.     

On the influence of mechanical conditions in osteochondral defect healing

Despite the introduction of new surgical techniques, the treatment of cartilage defects remains challenging. Delay or complete failure of cartilage healing is associated with problems in biological regeneration. The influence of mechanical conditions on this process, however, remains unevaluated. Osteochondral defects were generated on the left femoral condyle in 18 Yucatan minipigs. After 4, 6 and 12 weeks the defect filling, trabecular orientation and bone density were compared to the intact contralateral side. The mechanical straining during this period was then analyzed using an adaptive finite element technique. Histologically, the osteochondral defects showed bone resorption at the base and bone formation from the circumference. At 12 weeks, the macroscopically healed specimens showed fibrous cartilage formation, a minimally organized trabecular structure and increased trabecular volume fraction compared to the controls (p < 0.002). The amount of cancellous, cartilagineous, and fibrous tissue and the defect size as measured in histomorphometric analysis for the three time points (4, 6 and 12 weeks) was comparable in magnitude to that predicted by finite element analysis. The simulated osteochondral healing process was not fully capable of re-establishing a hyaline-like cartilage layer. The correlation between simulation and histology allows identification of mechanical factors that appear to have a larger impact on the healing of osteochondral defects than previously considered.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

Ca2+-modulated vision-linked ROS-GC guanylate cyclase transduction machinery

Vertebrate phototransduction depends on the reciprocal relationship between two-second messengers, cyclic GMP and Ca²⁺. The concentration of both is reciprocally regulated including the dynamic synthesis of cyclic GMP by a membrane bound guanylate cyclase. Different from hormone receptor guanylate cyclases, the cyclases operating in phototransduction are regulated by the intracellular Ca²⁺-concentration via small Ca²⁺-binding proteins. Based on the site of their expression and their Ca²⁺ modulation, this sub-branch of the cyclase family was named sensory guanylate cyclases, of which the retina specific forms are named ROS-GCs (rod outer segment guanylate cyclases). This review focuses on the structure and function of the ROS-GC subfamily present in the mammalian retinal neurons: photoreceptors and inner layers of the retinal neurons. Portions and excerpts of the review are from a previous chapter (Curr Top Biochem Res 6:111–144, 2004).