Initial characterization of a chlamydial receptor on mammalian cells

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.     

Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).     

The mechanisms of methionine oxidation concomitant with hormone radioiodination comparative studies of various oxidants using a simple new method

The mechanism of oxidation of methionine concomitant with iodination was studied by ascending paper chromatography using $\text{l}\text{-[Me-}{}^{14}\text{C}\text{]}\text{methionine}$. The ability of the principal iodination reactants to oxidize free methionine was measured in order to predict in situ methionyl oxidation during radioiodination of peptides. Iodide, oxidant, and tyrosine were tested individually and in combination. Dilute chloramine-T and H₂O₂ effectively oxidized free methionine, whereas electric current at the low levels used for iodination did not. Whereas KI₃ did not cause significant methionine oxidation, iodine in statu nascendi was a potent oxidant. Oxidation caused by chemical oxidants was markedly reduced by the addition of iodide, whereas the opposite effect was seen with electric current. In our in vitro system, tyrosine inhibited the effect of the chemical oxidants to different degrees, in the absence of iodide. Under conditions simulating actual ‘mild’ radioiodination conditions, with added tyrosine, none of the oxidants studied caused methionine oxidation; the presence of tyrosine and iodide appeared to preclude the oxidation of methionine. Both excess chloramine-T and high electric current resulted in the formation of a new compound, possibly a sulphinic acid derivative of methionine. The important finding was that the release of free iodine and its uptake by tyrosine were the dominant factors to be considered in the prevention of methionine oxidation.Our new chromatographic technique for the estimation of methionine oxidation in peptides is based on the observed similarity between the reactivity of free methionine and that of accessible methionyl residues in peptides. The method is simple, sensitive and reproducible.     

Radioimmunoassay of methionine5-enkephalin sulphoxide: Phylogenetic and anatomical distribution

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine⁵-enkephalin (Met⁵-Enk), Met⁵-Enk sulphoxide (Met⁵-Enk-S), has been developed. Antisera were raised in rabbits against Met⁵-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (¹²⁵I) Met⁵-Enk bound to antiserum by Met⁵-Enk was poor, but Met⁵-Enk-S displayed good displacement suggesting that the Met⁵-Enk immunogen was oxidised to Met⁵-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met⁵-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine⁵-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met⁵-Enk (2–5) sulphoxide and between 9% and 25% with D-Ala²-Met⁵-Enk sulphoxide. The antisera showed<0.01% cross-reactivity with other Met⁵-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met⁵-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met⁵-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met⁵-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met⁵-Enk, and may account for the relative insensitivity of some published RIAs using Met⁵-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met⁵-Enk IMR.     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).