Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.     

Continuous and long-term volume measurements with a commercial Coulter counter

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter.     

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.     

Characterization of Phosphate-Solubilizing Fluorescent Pseudomonads from the Rhizosphere of Seabuckthorn Growing in the Cold Deserts of Himalayas

Isolation and characterization of fluorescent pseudomonads with high phosphate-solubilizing ability is reported from the alkaline and calcium-rich soils with low P availability in the cold desert region of Lahaul and Spiti in the trans-Himalayas of India. Of 216 phosphate-solubilizing isolates, 12 exhibiting high solubilization of tricalcium phosphate (TCP) in NBRIP liquid culture were identified as Pseudomonas trivialis, P. poae, P. fluorescens, and Pseudomonas spp. on the basis of phenotypic features, whole-cell fatty acids methyl ester (FAME) profiles, and 16S rDNA sequencing. These isolates also showed relatively high solubilization of North Carolina rock phosphate (NCRP) in comparison to the solubilization of Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). The solubilization of phosphate substrates by P. trivialis and P. poae is reported for the first time.     

Regeneration and micrografting of lentil shoots

Summary A protocol for regeneration and micrografting of shoots of lentil (Lens culinaris Medik) was developed. Multiple shoots (4–5) were regenerated from cotyledonary node explants on Murashige and Skoog (MS) medium containing 8.8 μM 6-benzylaminopurine. In vitro regenerated shoots were micrografted on rootstocks with 96% efficiency. The successful grafts were transplanted to pots in Redi-earth^TM, hardened off and were grown to maturity with 100% success. The success of the micrografting was independent of the nature and concentration of growth regulator used in shoot initiation medium and the time period for induction of shoots. The protocol was successful with several cultivars of lentil. The advantages of micrografting over in vitro rooting are discussed.     

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Effect of ischemia-reperfusion injury on the morphology of peroxisomes

We have previously demonstrated that ischemic injury changed the density of peroxisomes into two distinct peaks, one with a normal density (1.21 g/cm³; Peak I) and a second peak with a lighter density (1. 14 g/cm³; Peak II).We studied the peroxisomes from both peaks under the Electron microscope. Examination of peak I following ischemia showed loss of matrix proteins and damaged limiting membranes with leakage of DAB positive material in direct proportion to the duration of ischemia. Upon reperfusion of the ischemic liver Peak I showed more severe damage to the organelle. These observations clearly demonstrated that ischemia reperfusion injury causes structural damage to peroxisomes. Interestingly ultrastructural examination of Peak II following ischemia showed evidence of perisomal proliferation with budding of existing peroxisomes and the presence of micro peroxisomes (changes similar to those noted under conditions leading to perisomal proliferation). However, peak II following reperfusion showed only damaged organelle. These observations underline the importance of peroxisomes in the response of the cell to ischemia-reperfusion injury.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.