Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24[emsp4 ]h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.     

Preselection of predatory mites to improve year-round biological control of western flower thrips in greenhouse crops

In spring and summer, two groups of natural enemies are successfully used for biological control of western flower thrips,Frankliniella occidentalis (Pergande) in greenhouses: phytoseiid mites (Amblyseius cucumeris (Oudemans) and, to a lesser extent,A. barkeri (Hughes)) and anthocorid bugs (Orius spp.). During winter, however, these predators often fail to control the pest. One likely cause for failure is the predators' tendency to enter diapause under short day conditions. In addition, eggs of predatory mites are generally susceptible to low humidity conditions, which often arise in greenhouses when outside temperatures drop below zero, or at bright, hot days in summer. In search for a thrips predator that is not hampered by these conditions, five subtropical phytoseiid species were selected which were known to feed on thrips:A. hibisci (Chant),A. degenerans Berlese,A. limonicus s.s. Garman and McGregor,A. scutalis (Athias-Henriot) andA. tularensis (Congdon). These species were compared toA. cucumeris andA. barkeri, with respect to the following features: (1) predation and oviposition rate with youngF. occidentalis larvae as prey, (2) oviposition rate on a diet of sweet pepper pollen, (3) drought tolerance spectrum of eggs, and (4) incidence of reproductive diapause under short day conditions. The results showed thatA. limonicus exhibited the highest predation and oviposition rates on a diet of thrips larvae. Moreover,A. limonicus females showed total absence of diapause under the conditions tested. A major disadvantage of this species was, however, that its eggs were most sensitive to low air humidity conditions. Least sensitive to low air humidity were eggs ofA. degenerans andA. hibisci. Females ofA. degenerans andA. hibisci also showed total absence of diapause, and intermediate rates of predation and oviposition, on both thrips larvae and pollen. In conclusion, we argue thatA. degenerans andA. hibisci are the most promising candidates for biological control ofF. occidentalis under conditions of low humidity and short day length. The success of these candidates remains to be shown in greenhouse experiments.     

Collection and characterization of amniotic fluid from scheduled C-section deliveries

Amniotic fluid (AF) possesses anti-inflammatory, anti-microbial and regenerative properties that make it attractive for use in clinical applications. The goals of this study were to assess the feasibility of collecting AF from full-term pregnancies and to evaluate non-cellular and cellular properties of AF for clinical applications. Donor informed consent and medical histories were obtained from pregnant women scheduled for C-sections and infectious disease testing was performed the day of collection. AFs were evaluated for total volume, fluid chemistries, total protein, and hyaluronic acid (HA) levels. AF was also assessed with quantitative antibody arrays, cellular content and for an ability to support angiogenesis. Thirty-six pregnant women consented and passed donor screening to give birth tissue. AF was successfully collected from 17 individuals. Median AF volumes were 70 mL (range 10–815 mL; n = 17). Fluid chemistries were similar, but some differences were noted in HA levels and cytokine profiles. Cytokine arrays revealed that an average of 304 ± 20 of 400 proteins tested were present in AF with a majority of cytokines associated with host defense. AF supported angiogenesis. Epithelioid cells were the major cell type in AF with only a minor population of lymphoid cells. Cultures revealed a highly proliferative population of adherent cells capable of producing therapeutic doses of mesenchymal stromal cells (MSCs). These findings showed that significant volumes of AF were routinely collected from full-term births. AF contained a number of bioactive proteins and only a rare population of MSCs. Variations noted in components present in different AFs, warrant further investigations to determine their relevance for specific clinical applications.     

Comprehensive Reference Ranges for Hematology and Clinical Chemistry Laboratory Parameters Derived from Normal Nigerian Adults

Background

Interpretation of laboratory test results with appropriate diagnostic accuracy requires reference or cutoff values. This study is a comprehensive determination of reference values for hematology and clinical chemistry in apparently healthy voluntary non-remunerated blood donors and pregnant women.

Methods and findings

Consented clients were clinically screened and counseled before testing for HIV, Hepatitis B, Hepatitis C and Syphilis. Standard national blood donors’ questionnaire was administered to consented blood donors. Blood from qualified volunteers was used for measurement of complete hematology and chemistry parameters. Blood samples were analyzed from a total of 383 participants, 124 (32.4%) males, 125 (32.6%) non-pregnant females and 134 pregnant females (35.2%) with a mean age of 31 years. Our results showed that the red blood cells count (RBC), Hemoglobin (HB) and Hematocrit (HCT) had significant gender difference (p = 0.000) but not for total white blood count (p>0.05) which was only significantly higher in pregnant verses non-pregnant women (p = 0.000). Hemoglobin and Hematocrit values were lower in pregnancy (P = 0.000). Platelets were significantly higher in females than men (p = 0.001) but lower in pregnant women (p = 0.001) with marked difference in gestational period. For clinical chemistry parameters, there was no significant difference for sodium, potassium and chloride (p>0.05) but gender difference exists for Bicarbonate (HCO3), Urea nitrogen, Creatinine as well as the lipids (p<0.05). Total bilirubin was significantly higher in males than females (p = 0.000). Significant differences exist for all chemistry parameters between pregnant and non-pregnant women in this study (p<0.05), except Amylase and total cholesterol (p>0.05).

Conclusions

Hematological and Clinical Chemistry reference ranges established in this study showed significant gender differences. Pregnant women also differed from non-pregnant females and during pregnancy. This is the first of such comprehensive study to establish reference values among adult Nigerians and difference observed underscore the need to establish reference values for different populations.     

Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac,ADAM17/TACE,and RhoA in tubular epithelial cells

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α–induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration.     

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase

The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7 kDa–11.5 kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the β′ subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always β′, many interactions are taxon‐specific. ω‐less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the β′ subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω‐less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω‐less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.     

Structure and function of a cyanophage-encoded peptide deformylase

Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.     

Triazolopyridazine LRRK2 kinase inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson’s disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of [1,2,4]triazolo[4,3-b]pyridazines that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays and show an unprecedented selectivity towards the G2019S mutant. A structural rational for the observed selectivity is proposed.