Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Ultrastructural evidence of oestradiol receptor by immunochemistry

Antiserum against calf uterus oestradiol receptor has been used for detecting oestradiol receptor in rat pituitary cells at the ultrastructural level after immunochemical reaction according to Sternberger. The gonadotropic, lactotropic and somatotropic cells were positive, but not the thyrotropic and corticotropic cells. In peripubertal and adult rats, both cytoplasmic and nuclear receptors were seen, but in a long-term castrated rat, the receptor was found only in the cytoplasm. After oestradiol administration to 21-day-old animals, the cytoplasmic receptor decreased and the nuclear receptor increased in gonadotropic cells, supporting the concept of hormone-receptor complex translocation. Antibodies against α1-foetoprotein demonstrated the presence of this oestrogen-binding plasma protein in all pituitary cells, but only in the cytoplasmic area. These results and the immunological controls related to antibody specificity give the first evidence of steroid receptor at the ultrastructural level.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

The blacks of Panama: their genetic diversity as assessed by 15 inherited biochemical systems

Panama's black citizens are culturally and historically divisible into two groups, the Spanish-speaking coloniales and the English-speaking anglos or afro-antillanos. Until recently these groups have been geographically as well as culturally isolated one from the other, although both are predominantly of West and Southwest African origin. Assessment of the genetic diversity within-villages and within language groups reveals as much, possibly somewhat more, diversity in 15 inherited biochemical markers within villages and language groups as that which obtains between villages and language groups. A number of rare variants at the 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and esterase D loci were encountered and are described.     

A Chemical Proteomics Approach for the Search of Pharmacological Targets of the Antimalarial Clinical Candidate Albitiazolium in Plasmodium falciparum Using Photocrosslinking and Click Chemistry

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug–interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug–interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets.