Acylation of hemoglobin by succinyldisalicylate, a potential crosslinking reagent.

Diaspirin, HO₂CC₆H₄OOCCH₂CH₂COOC₆H₄CO₂H, acylates hemoglobin more effectively than does aspirin. The disalicylate has the potential to act as a crosslinking reagent in proteins and other molecules in general.     

Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization.

Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.     

Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.     

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A₁, A_2A and A₃ adenosine receptors and in adenylyl cyclase experiments for their potency at the human A_2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N ⁶ position of 9-propyladenine significantly increased binding affinity at the human A₁ and A₃ adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N ⁶ position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A_2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A_2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.     

Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature

Plasmodium undergoes one round of multiplication in the liver prior to invading erythrocytes and initiating the symptomatic blood phase of the malaria infection. Productive hepatocyte infection by sporozoites leads to the generation of thousands of merozoites capable of erythrocyte invasion. Merozoites are released from infected hepatocytes as merosomes, packets of hundreds of parasites surrounded by host cell membrane. Intravital microscopy of green fluorescent protein-expressing P. yoelii parasites showed that the majority of merosomes exit the liver intact, adapt a relatively uniform size of 12-18 microm, and contain 100-200 merozoites. Merosomes survived the subsequent passage through the right heart undamaged and accumulated in the lungs. Merosomes were absent from blood harvested from the left ventricle and from tail vein blood, indicating that the lungs effectively cleared the blood from all large parasite aggregates. Accordingly, merosomes were not detectable in major organs such as brain, kidney, and spleen. The failure of annexin V to label merosomes collected from hepatic effluent indicates that phosphatidylserine is not exposed on the surface of the merosome membrane suggesting the infected hepatocyte did not undergo apoptosis prior to merosome release. Merosomal merozoites continued to express green fluorescent protein and did not incorporate propidium iodide or YO-PRO-1 indicating parasite viability and an intact merosome membrane. Evidence of merosomal merozoite infectivity was provided by hepatic effluent containing merosomes being significantly more infective than blood with an identical low-level parasitemia. Ex vivo analysis showed that merosomes eventually disintegrate inside pulmonary capillaries, thus liberating merozoites into the bloodstream. We conclude that merosome packaging protects hepatic merozoites from phagocytic attack by sinusoidal Kupffer cells, and that release into the lung microvasculature enhances the chance of successful erythrocyte invasion. We believe this previously unknown part of the plasmodial life cycle ensures an effective transition from the liver to the blood phase of the malaria infection.     

An helicoidal structure surrounding the cilium axoneme: visualization by the monoclonal antibody CC-248

Monoclonal antibody CC-248 labels cilia differentially on Triton X-100 permeabilized ciliated epithelium of quail oviduct by indirect immunofluorescence. On isolated ciliated cells, a punctuated staining is seen at the distal region over the bend of cilia. Electron micrographs of immunoperoxidase and immunogold techniques showed that the punctuated fluorescence corresponds to a helical disposition of CC-248 antigenic sites. This labeling was arranged on the axonemal distal region either as a simple or a double helix externally disposed around the nine microtubular doublets. These results suggest the existence of a detergent insoluble structure in the ciliary matrix that might concern the ciliary skeleton, probably acting as an elastic recoil that keeps the structural integrity of the axoneme during bending. The cross-reactivity of CC-248 MAb with the intermediate filament cytoskeleton of ciliated and smooth muscle cells indicates that this structure might be related to the intermediate filament family.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.     

Identification of the binding sites of the SR49059 nonpeptide antagonist into the V1a vasopressin receptor using sulfydryl-reactive ligands and cysteine mutants as chemical sensors

To identify the binding site of the human V1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or alpha-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family.