Joenel Alcantara

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment.

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.

Plant Defense Responses in Opium Poppy Cell Cultures Revealed by Liquid Chromatography-Tandem Mass Spectrometry Proteomics

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.

The region of human transferrin involved in binding to bacterial transferrin receptors is iocalized in the C‐lobe

Iron‐saturated human transferrin was digested with either chymotrypsin or trypsin to produce C‐lobe and N‐lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C‐lobe and N‐lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C‐lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin‐binding proteins (Tbps), we demonstrated that both transferrin‐binding proteins were able to bind to the C‐lobe fragment of human transferrin.

The structural basis of transferrin sequestration by transferrin-binding protein B

Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin during infection through a surface transferrin receptor system composed of proteins TbpA and TbpB. Here we present the crystal structures of TbpB from N. meningitidis in its apo form and in complex with human transferrin. The structure reveals how TbpB sequesters and initiates iron release from human transferrin.

Evidence for non-siderophore-mediated acquisition of transferrin-bound iron by Pasteurella multocida

Two clinical isolates of Pasteurella multocida associated with bovine pneumonia were examined for iron acquisition. Both isolates were capable of obtaining iron for growth from bovine but not from human, avian, equine or porcine transferrin. This correlated with specific binding of bovine transferrin by iron-limited cells or isolated membranes. No siderophore was detected in the strains by a general screening assay. In response to iron-limited conditions, a number of high molecular mass iron-regulated outer membrane proteins were produced including an 82 kDa receptor protein which was affinity isolated with biotinylated transferrin. In contrast, avian strains of P. multocida could not use transferrin-bound for growth and did not express either transferrin binding activity or the 82 kDa receptor protein.

Evaluation of meningococcal serogroup C conjugate vaccine programs in Canadian children: interim analysis.

BACKGROUND To assess antibody titers afforded by meningococcal C- (MenC) tetanus toxoid conjugate vaccine at 12 months of age in three different immunization schedules. METHODS This prospective study included three similar cohorts of healthy infants from 1-dose, 2-dose and 3-dose MenC infant immunization programs. Infants were enrolled at 12 months of age and given the final scheduled dose of MenC-tetanus toxoid conjugate vaccine with sera collected prior to and 1 month after the vaccination. Serum bactericidal activity (SBA) titers ≥ 1:8 were considered protective. RESULTS Before the 12 month dose, participants had significantly different protective titers according to the number of prior doses received: 100% (95% CI 97.6-100%) of infants who had 2 prior doses (at 2 and 4 months) were protected compared to 84.0% (76.7-89.3%) of participants with one dose (at 2 months) and 27.6% (21.0-35.4%) of unvaccinated infants. All subjects were protected after the 12 month MenC dose, but titers were higher with prior priming. CONCLUSIONS Two MenC doses given in infancy afford optimal protection during the first year of life; however, substantial protection was seen after one dose at 2 months.

Prevalence and impact of Streptococcus pneumoniae in adult cystic fibrosis patients: a retrospective chart review and capsular serotyping study

BackgroundCystic fibrosis (CF) is a genetic disease characterized by complex polymicrobial communities within the lower respiratory tract. S. pneumoniae, while a well-defined pathogen in the general population, has rarely been identified in CF. Furthermore, prevalence studies on Pneumococcus in CF have predominantly focused on the infant and pediatric populations, and outcome data is lacking.MethodsThrough a review of our comprehensive clinical and microbiologic database from a single adult CF center in Canada from 1978–2013 we sought to determine the incidence, prevalence, serotype and clinical impact of Pneumococcus in adults with CF.ResultsOnly fifteen of 318 adult CF patients (5%) were ever found to have transient Pneumococcus colonization, and none developed persistent infection although length of carriage varied. As all isolates were stored, capsular serotyping could be performed using a multiplex PCR panel. Capsular serotyping revealed a varied distribution of several serotypes within these isolates. Lung function testing at time of incident Pneumococcus isolation was compared with values before and after isolation and showed no significant reduction in spirometry values, nor was there an increased need for rescue antibacterial therapy.ConclusionWithin our center, incident Pneumococcus infection is neither common, associated with a disproportionate clinical deterioration nor results in chronic infection.

PROKARYO: an illustrative and interactive computational model of the lactose operon in the bacterium Escherichia coli

BackgroundWe are creating software for agent-based simulation and visualization of bio-molecular processes in bacterial and eukaryotic cells. As a first example, we have built a 3-dimensional, interactive computer model of an Escherichia coli bacterium and its associated biomolecular processes. Our illustrative model focuses on the gene regulatory processes that control the expression of genes involved in the lactose operon. Prokaryo, our agent-based cell simulator, incorporates cellular structures, such as plasma membranes and cytoplasm, as well as elements of the molecular machinery, including RNA polymerase, messenger RNA, lactose permease, and ribosomes.ResultsThe dynamics of cellular ’agents’ are defined by their rules of interaction, implemented as finite state machines. The agents are embedded within a 3-dimensional virtual environment with simulated physical and electrochemical properties. The hybrid model is driven by a combination of (1) mathematical equations (DEQs) to capture higher-scale phenomena and (2) agent-based rules to implement localized interactions among a small number of molecular elements. Consequently, our model is able to capture phenomena across multiple spatial scales, from changing concentration gradients to one-on-one molecular interactions.We use the classic gene regulatory mechanism of the lactose operon to demonstrate our model’s resolution, visual presentation, and real-time interactivity. Our agent-based model expands on a sophisticated mathematical E. coli metabolism model, through which we highlight our model’s scientific validity.ConclusionWe believe that through illustration and interactive exploratory learning a model system like Prokaryo can enhance the general understanding and perception of biomolecular processes. Our agent-DEQ hybrid modeling approach can also be of value to conceptualize, illustrate, and—eventually—validate cell experiments in the wet lab.

Do Dose Numbers Matter? Evaluation of Differing Infant and Toddler Meningococcal C Conjugate Vaccine Programs in Canadian Children.

Background: The diversity of Canadian infant meningococcal C conjugate (MenC) vaccine programs is unique among countries providing MenC vaccines and offers a valuable opportunity to determine the optimal vaccine program. This longitudinal study assessed differences in seroprotection by 3 different vaccine schedules in children two years after receiving either 1 toddler MenC vaccine dose (1 dose), 1 infant and 1 toddler dose (2 doses), or 2 infant and 1 toddler MenC vaccine dose (3 doses). Methods: Three similar cohorts of healthy infants from 1, 2 and 3 dose program areas were enrolled before to their 12 month toddler dose and vaccinated with MenC-tetanus toxoid (MenC-TT) conjugate vaccine. Sera obtained 2 years later were assayed for serogroup C bactericidal activity using standardized procedures with rabbit as the exogenous complement source. Serum bactericidal activity titers ≥1:8 were considered protective. Results: Results were available for 384 children. Rates of seroprotection at 36 months of age were significantly different between the 1 and 3 dose programs, but confidence intervals overlapped between the 1 and 2 dose programs and between the 2 and 3 dose programs: 1 dose 92% (95% confidence interval: 86%–96%) versus 99% (95%–100%) with 2 doses and 100% (97%–100%) with 3 doses. Geometric mean titers were significantly different at 12.1 (10.8–13.5), 32.4 (28.9–36.2) and 50.6 (45.7–55.9) in the 1, 2 and 3 dose programs, respectively. Conclusions: At 36 months of age, evidence of seroprotection remained for greater than 90% of participants. Our results indicate that 1 toddler dose or 1 infant plus 1 toddler dose with MenC-TT vaccine provides seroprotection against MenC disease in early childhood.