Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

Glycine significantly enhances bacterial membrane vesicle production: a powerful approach for isolation of LPS-reduced membrane vesicles of probiotic Escherichia coli

Bacterial membrane vesicles (MVs) have attracted strong interest in recent years as novel nanoparticle delivery platforms. Glycine is known to induce morphological changes in the outer layer of bacteria. We report here that glycine dramatically facilitates MV production in a flagella-deficient mutant of the non-pathogenic probiotic Escherichia coli strain Nissle 1917. Supplementation of culture medium with 1.0% glycine induced cell deformation at the early exponential phase, eventually followed by quasi-lysis during the late exponential to stationary phase. Glycine supplementation also significantly increased the number of MVs with enlarged particle size and altered the protein profile with an increase in the inner membrane and cytoplasmic protein contents as compared to non-induced MVs. Of note, the endotoxin activity of glycine-induced MVs was approximately eightfold or sixfold lower than that of non-induced MVs when compared at equal protein or lipid concentrations respectively. Nevertheless, glycine-induced MVs efficiently induced both immune responses in a mouse macrophage-like cell line and adjuvanticity in an intranasal vaccine mouse model, comparable to those of non-induced MVs. We propose that the present method of inducing MV production with glycine can be used for emerging biotechnological applications of MVs that have immunomodulatory activities, while dramatically reducing the presence of endotoxins.     

Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.     

XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.     

Relationships of anti-PAc (361-386) peptide salivary IgA antibody, eosinophils and basophils with periodontal status in the elderly

The amino acid residues 361-386 of Streptococcus mutans PAc includes an important region associated with the interaction between S. mutans and salivary components. We investigated the relationships between levels of the anti-PAc (361-386) peptide antibody (PPA) in saliva and periodontal status in 281 elderly subjects (mean age 77 years; 118 females, 163 males) by assessing dental calculus (CA), attachment loss (AL), pocket depth (PD), bleeding on probing (BOP) and various blood parameters. Enzyme-linked immunosorbent assay results revealed that subjects with a PPA level of greater than 0.1 (PPA detected group) showed a lower average value for number of sites with more than 6 mm of AL/6 points x 100/tooth (rAL6) than those with a PPA level of less than 0.1 (PPA not detected group). Furthermore, average values for rAL6 were significantly lower in the PPA detected group, and BOP, AL and rAL6 correlated positively and significantly with the percentage of eosinophils present in leukocytes in female subjects in both groups. PPA level had a negative correlation with percentages of basophils and eosinophils. The results indicate that systemic increases in numbers of eosinophils and basophils are associated with the development of periodontal diseases, while PPA level may be a useful indicator of periodontal status.     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

Split introns in the genome of Giardia intestinalis are excised by spliceosome-mediated trans-splicing

Spliceosomal introns are hallmarks of most eukaryotic genomes and are excised from premature mRNAs by a spliceosome that is among the largest, and most complex, molecular machine in cells. The divergent unicellular eukaryote Giardia intestinalis, the causative agent of giardiasis, also possesses spliceosomes, but only four canonical (cis-spliced) introns have been identified in its genome to date. We demonstrate that this organism has a novel form of spliceosome-mediated trans-splicing of split introns that is essential for generating mature mRNAs for at least two important genes: one encoding a heat shock protein 90 (HSP90), which controls the conformation of a suite of cellular proteins, and the other encoding a dynein molecular motor protein, involved in the motility of eukaryotic flagella. These split introns have properties that distinguish them from other trans-splicing systems known within eukaryotes, suggesting that Giardia independently evolved a unique system to splice split introns.     

Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response

We previously reported that mutation of galE in Porphyromonas gingivalis has pleiotropic effects, including a truncated lipopolysaccharide (LPS) O-antigen and deglycosylation of the outer membrane protein OMP85 homolog. In the present study, further analysis of the galE mutant revealed that it produced little or no outer membrane vesicles (OMVs). Using three mouse antisera raised against whole cells of the P. gingivalis wild type strain, we performed ELISAs to examine the reactivity of these antisera with whole cells of the wild type or the galE mutant. All three antisera had significantly lower reactivity against the galE mutant compared to wild type. OMVs, but not LPS, retained the immunodominant determinant of P. gingivalis, as determined by ELISAs (with wild type LPS or OMVs as antigen) and absorption assays. In addition, we assessed the capacity of OMVs as a vaccine antigen by intranasal immunization to BALB/c mice. Synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [Poly (I∶C)], an agonist of Toll-like receptor 3 (TLR3), was used as the mucosal adjuvant. Vaccination with OMV elicited dramatically high levels of P. gingivalis-specific IgA in nasal washes and saliva, as well as serum IgG and IgA. In conclusion, the OMVs of P. gingivalis have an important role in mucosal immunogenicity as well as in antigenicity. We propose that P. gingivalis OMV is an intriguing immunogen for development of a periodontal disease vaccine.