The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance

Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.     

Stem cells, regenerative medicine, and animal models of disease

The field of stem cell biology and regenerative medicine is rapidly moving toward translation to clinical practice, and in doing so has become even more dependent on animal donors and hosts for generating cellular reagents and assaying their potential therapeutic efficacy in models of human disease. Advances in cell culture technologies have revealed a remarkable plasticity of stem cells from embryonic and adult tissues, and transplantation models are now needed to test the ability of these cells to protect at-risk cells and replace cells lost to injury or disease. With such a mandate, issues related to acceptable sources and controversial (e.g., chimeric) models have challenged the field to provide justification of their potential efficacy before the passage of new restrictions that may curb anticipated breakthroughs. Progress from the use of both in vitro and in vivo regenerative medicine models already offers hope both for the facilitation of stem cell phenotyping in recursive gene expression profile models and for the use of stem cells as powerful new therapeutic reagents for cancer, stroke, Parkinson's, and other challenging human diseases that result in movement disorders. This article describes research in support of the following three objectives: (1) To discover the best stem or progenitor cell in vitro protocols for isolating, expanding, and priming these cells to facilitate their massive propagation into just the right type of neuronal precursor cell for protection or replacement protocols for brain injury or disease, including those that affect movement such as Parkinson's disease and stroke; (2) To discover biogenic factors--compounds that affect stem/progenitor cells (e.g., from high-throughput screening and other bioassay approaches)--that will encourage reactive cell genesis, survival, selected differentiation, and restoration of connectivity in central nervous system movement and other disorders; and (3) To establish the best animal models of human disease and injury, using both small and large animals, for testing new regenerative medicine therapeutics.     

Eucalyptus has functional equivalents of the Arabidopsis AP1 gene

Two Eucalyptus homologues of the Arabidopsis floral homeotic gene AP1 (EAP1 and EAP2/) show 60–65% homology to AP1. EAP1 and EAP2 are expressed predominantly in flower buds. EAP2 produces two different polypeptides arising from differential splicing at an intron, the shorter EAP2 protein diverging from the longer sequence after amino acid 197 and having a translation stop after residue 206. This truncated protein includes both MADS- and K-box amino acid sequences. Ectopic expression of the EAP1 or either of the two EAP2 polypeptides in Arabidopsis driven by the 35S promoter produces effects similar to the corresponding AP1 construct, causing plants to flower earlier, have shorter bolts and resemble the terminal flower mutant (tfl).     

Analysis of helicase gene mutations in Japanese Werner’s syndrome patients

The profile of helicase gene mutations was studied in 89 Japanese Werner’s syndrome (WRN) patients by examining the previously described mutations 1– 4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotype analysis of the markers surrounding the WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders. Received: 25 July 1996 / Revised: 20 September 1996     

Parasite influences on host life history: Echinostoma revolutum parasitism of Lymnaea elodes snails

Using field surveys and experimental infections, we investigated the influence of a trematode parasite on life history traits of adult Lymnaea elodes snails. We found that parasitism significantly affected the growth, fecundity, and survival of host snails. Within five of the six natural L. elodes populations we sampled, shell length of echinostome-infected hosts was significantly greater than for uninfected conspecifics. Furthermore, we show that gigantism occurs among experimentally infected snails due to an accelerated growth rate and size-selective mortality following an Echinostoma revolutum infection. The fecundity of infected snails sharply decreased beginning at 3 weeks post exposure (PE) and all egg production eventually ceased for most hosts by 5–6 weeks PE. Energy constraints, imposed by parasite development, alter the host energy budget. Early in the infection, parasite depletion of host energy reserves reduces host reproduction, but sufficient resources remain to allow accelerated host growth. Mortality was increased among host snails at two distinct stages: shortly after exposure and several weeks after cercariae were first released. We did not observe tissue degradation in snails during the first 4 weeks after exposure to the parasite, but destruction of host tissues was noted among snails dying later in the infection. Received: 5 September 1997 / Accepted: 19 November 1997     

Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents

Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most potent analogs are the 5-(N-methyl, N-ethyl, and N-methylazetidine methyl) piperidyl derivatives 12, 23, and 34. These compounds have a broad spectrum of activity. Based on the in vivo efficacy and cost of synthesis, the N-ethyl analog 23 was chosen as a novel anticoccidial agent for a field trial.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles

The in vivo behavior of shell cross-linked knedel-like (SCK) nanoparticles is shown to be tunable via a straightforward and versatile process that advances SCKs as attractive nanoscale carriers in the field of nanomedicine. Tuning of the pharmacokinetics was accomplished by grafting varied numbers of methoxy-terminated poly(ethylene glycol) (mPEG) chains to the amphiphilic block copolymer precursors, together with chelators for the radioactive tracer and therapeutic agent (64)Cu, followed by self-assembly into block copolymer micelles and chemical cross-linking throughout the shell regions. (64)Cu-radiolabeling was then performed to evaluate the SCKs in vivo by means of biodistribution experiments and positron emission tomography (PET). It was found that the blood retention of PEGylated SCKs could be tuned, depending on the mPEG grafting density and the nanoparticle surface properties. A semiquantitative model of the density of mPEG surface coverage as a function of in vivo behavior was applied to enhance the understanding of this system.