Expression analysis of Fgf8a & Fgf8b in early stage of P19 cells during neural differentiation

Fgf8 is a member of the fibroblast growth factor (FGF) family that plays an important role in early neural development. Cellular aggregation and retinoic acid (RA) are needed for mouse embryonic carcinoma (EC) P19 cell neural differentiation. We have examined the Fgf8 gene in P19 cells during neural differentiation and identified 2 alternatively spliced Fgf8 isoforms, Fgf8a and Fgf8b, among the 8 known splicing isoforms in mammals. The expression of Fgf8a and Fgf8b mRNAs transiently and rapidly increased in the early stage of P19 cells during RA-induced neural differentiation, followed by a decline in expression. The relative amount of Fgf8b was clearly higher than that of Fgf8a at different time-points measured within 24 h after RA treatment. Increased Fgf8b mRNA expression was cellular-aggregation dependent. The results demonstrated that cellular-aggregation-induced Fgf8b, but not Fgf8a, may play a pivotal role in early neural differentiation of P19 cells.     

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K_M^DXP = 104 µM, K_M^NADPH = 13 µM, k_cat^DXP = 2 s⁻¹, k_cat^NADPH = 1.3 s⁻¹), an IC₅₀ for fosmidomycin of 247 nM, and a K_i for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg⁺² as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.     

Three-dimensional structure prediction of bovine AP lyase, BAP1: prediction of interaction with DNA and alterations as a result of Arg176-->Ala, Asp282-->Ala, and His308-->Asn mutations

BAP1 is an apurinic/apyrimidinic lyase (AP lyase) that plays an important role in the repair of DNA damage. The present study deals with the prediction of the 3D structure of bovine AP lyase based on its sequence homology with human AP lyase. The predicted 3D model of bovine AP1 shows remarkable similarity with human endonuclease in the overall 3D fold. However, significant differences in the model and the X-ray structure were located at some of the important sites. We have analyzed the active center of the enzyme and other sites that are involved in DNA repair. A number of amino acids bind the bases located in the major/minor grooves of DNA. An insertion of Arg176 in the major groove and Met270 in the minor groove caps the DNA bound enzyme's active site, stabilizing the extra helical AP site conformation and effectively locking the protein onto the AP-DNA. Three BAP1 mutants were also modeled and analyzed as regards the changes in the structure. Substitution of Arg176-->Ala leads to the loss of DNA binding whereas mutation of Asp282-->Ala and His308-->Asn leads to a decrease in the enzymatic activity.     

An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections

The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.     

Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes

Intracellular collagen degradation in normal rat hepatocytes was exponetially stimulated by db-cAMP (10–100 µM). The effect was manifested as a decrease (p < 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only upto a threshold concentration (16.2 ± 1.3 p moles/10⁶ cells) elicited by 100 µM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10–50 µM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p < 0.05) in hepatocytes from rats administered CCL₄. However, the extent of stimulation was significantly (p < 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.Abbreviations cAMP 3,5 cyclic Adenosine Monophosphate - db-cAMP dibutyryl cyclic Adenosine Monophosphate - TCA Trichloroacetic Acid - Coll. Collagen - DMEM Dulbecoo's Minimal Essential Medium     

Bismuth(III) deferiprone effectively inhibits growth of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> ATCC 27774

Sulfate-reducing bacteria have been implicated in inflammatory bowel diseases and ulcerative colitis in humans and there is an interest in inhibiting the growth of these sulfide-producing bacteria. This research explores the use of several chelators of bismuth to determine the most effective chelator to inhibit the growth of sulfate-reducing bacteria. For our studies, Desulfovibrio desulfuricans ATCC 27774 was grown with nitrate as the electron acceptor and chelated bismuth compounds were added to test for inhibition of growth. Varying levels of inhibition were attributed to bismuth chelated with subsalicylate or citrate but the most effective inhibition of growth by D. desulfuricans was with bismuth chelated by deferiprone, 3-hydroxy-1,2-dimethyl-4(1H)-pyridone. Growth of D. desulfuricans was inhibited by 10 μM bismuth as deferiprone:bismuth with either nitrate or sulfate respiration. Our studies indicate deferiprone:bismuth has bacteriostatic activity on D. desulfuricans because the inhibition can be reversed following exposure to 1 mM bismuth for 1 h at 32 °C. We suggest that deferiprone is an appropriate chelator for bismuth to control growth of sulfate-reducing bacteria because deferiprone is relatively nontoxic to animals, including humans, and has been used for many years to bind Fe(III) in the treatment of β-thalassemia.