Cortical State Fluctuations across Layers of V1 during Visual Spatial Perception

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Trio Haploinsufficiency Causes Neurodevelopmental Disease-Associated Deficits

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Design and synthesis of paclitaxel conjugated with an ErbB2-recognizing peptide, EC-1

The selective delivery of therapeutic agents to receptors overexpressed in cancer cells without harming the rest of the body is a major challenge in clinical oncology today. In this study, we report the design and synthesis of paclitaxel (PTX) conjugated with an erbB2-recognizing peptide (EC-1). The cyclic peptide EC-1 specifically binds to the extracellular domain of ErbB2 and selectively inhibits proliferation of breast cancer cells overexpressing ErbB2. PTX is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer, yet patients have to suffer some side effects caused by its systemic toxicity. The aim of our conjugate is to specifically deliver antitumor agent PTX to breast cancer cells that overexpress oncogenic ErbB2 with the purpose to reduce toxicity and enhance selective killing of cancer cells. In this study, a concise and efficient synthetic route for the preparation of the PTX-EC-1 conjugate has been developed in 6% overall yield. This synthetic approach provides a general method for conjugating a highly functionalized and disulfide-bridge containing cyclopeptide to Taxol or other antitumor agents.     

Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain

Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Decreased pCO(2) accumulation by eliminating bicarbonate addition to high cell-density cultures

High-density perfusion cultivation of mammalian cells can result in elevated bioreactor CO(2) partial pressure (pCO(2)), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 x 10(6) cells/mL, the bioreactor pCO(2) exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO(3), and 40% from NaHCO(3) added for pH control. Recognizing the limitations to the practicality of gas sparging for CO(2) removal in perfusion systems, a strategy based on CO(2) reduction at the source was investigated. The NaHCO(3) in the medium was replaced with a MOPS-Histidine buffer, while Na(2)CO(3) replaced NaHCO(3) for pH control. These changes resulted in 63-70% pCO(2) reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing-scale. Bioreactor pCO(2) values after these modifications were in the 68-85 mm Hg range, pCO(2) reductions consistent with those theoretically expected. Low bioreactor pCO(2) was accompanied by both 68-123% increased growth rates and 58-92% increased specific productivity. Bioreactor pCO(2) reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO(2) reduction at the source through medium and base modification should be readily applicable to large-scale fed-batch cultivation of mammalian cells.     

Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles

Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here, we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately two- to three-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity.     

Cancer risk assessment for 1,3-butadiene: dose-response modeling from an epidemiological perspective

The dose-response assessment of the association between 1,3-butadiene (BD) and leukemia mortality among workers in the North American synthetic rubber industry is explored. Analyses are based on the most recent University of Alabama at Birmingham epidemiological study and exposure estimation. The U.S. EPA Science Advisory Board recommendations of using the most recent data and giving consideration to peak exposures to BD have been followed. If cumulative BD ppm-years is to be used as the predictor of the leukemia rate ratio, then the performance of that predictor is statistically significantly improved if the slope in the predictor is estimated with age and the cumulative number of BD peaks (where a BD peak is any exposure, regardless of duration, to a BD concentration above 100 ppm) added as categorical covariates. After age and the cumulative number of BD peaks are incorporated as categorical covariates in the Poisson regression model, the estimated concentration (EC(001)) corresponding to an excess risk of 0.001 as a result of continuous environmental exposure is 11.2 ppm; however, the estimated slope for BD cumulative ppm-years in the linear rate ratio for leukemia used to derive this EC(001) is not statistically significantly different from zero. Sensitivity analyses using alternative models indicate either essentially no risk or estimated EC(001) values of 9 and 77 ppm. Analyses suggesting the absence of a statistically significant low-dose risk versus cumulative BD ppm-years are presented. Sensitivity analyses of other malignant neoplasms of lymphatic and hematopoietic tissue (specifically, lymphoid and myeloid neoplasms) resulted in conclusions about the dose-response modeling methodology that were supportive of the methodology used for leukemia.     

TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.     

Leishmania (Viannia) braziliensis: epidemiology of canine cutaneous leishmaniasis in the State of Paraná (Brazil)

The present study examines the role that dogs play in the maintenance of the Leishmania cycle in the State of Paraná, Southern Brazil. Dogs were examined in three regions where cutaneous leishmaniasis is endemic or epidemic (R1-Vale da Ribeira; R2-Central region of Paraná State and R3-Northern region). To determine serum prevalence rates ELISA was used. In regions endemic for Trypanosoma cruzi (R1 and R3), serum from dogs seroreactive towards Leishmania antigen was subjected to T. cruzi adsorption in order to eliminate cross-reaction with common antigen epitopes. Concomitantly, dogs with cutaneous lesions were biopsied to isolate and identify parasites using RAPD. Leishmania were classified by the phenetic method using the Jaccard coefficient of similarity, and grouped by Unweighted Pair-Group Method using an Arithmetic Average (UPGMA). A total of 410 dogs were studied. In R1 (Vale da Ribeira) 159 dogs were evaluated of which 10 had anti-Leishmania antibody. In R2 (Central Paraná), 39 animals were examined of which 8 were seropositive. In R3 (the North) 212 dogs were evaluated of which 39 animals were seropositive. Thirteen dogs had cutaneous lesions and the parasites were isolated from a dog with mucocutaneous lesion in R1, two animals with simple skin lesions in R2 and 10 dogs with multiple lesions in R3. The identification of the parasite by molecular methods showed it to be L. (Viannia) braziliensis. Based on this information, the role of domestic dogs in Leishmania infection of cutaneous leishmaniasis in Paraná is discussed.