Alterations in the protein-synthesis, -degradation and/or -secretion rates in hepatic subcellular fractions of selenium-deficient mice.

Conformational studies of myelin basic protein (MBP) in solution generally have used protein purified in organic solvents and acid. The use of such conditions raises the possibility that the secondary structure reported for the basic protein represents a denatured state. Therefore we have purified this protein by using a procedure that avoids denaturants. Bovine myelin was extracted with 0.2 M-CaCl2 and the protein was purified from the supernatant by chromatography on Sephadex G-75. The conformation of the basic protein was characterized by using c.d. and 1H-n.m.r. spectroscopy. In solution, it appeared to be predominantly randomly coiled, with only small segments of persistent structure. However, in the presence of myristoyl lysophosphatidylcholine the secondary structure of MBP became more ordered, and sedimentation-velocity experiments showed that MBP aggregated. Comparison of our results with published data indicates that Ca2+-extracted basic protein behaves similarly to the protein purified by traditional methods with respect to its ordered conformation in solution in the absence and in the presence of lipid and with respect to its self-association. Thus its thermodynamically stable structure in aqueous solution appears to be a highly flexible coil.     

Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.     

Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.     

Expression of the first calcitonin/CGRP gene in spontaneous and transplanted rat medullary thyroid carcinoma: a comparison of dot-blot analysis, in situ hybridization, and immunohistochemistry.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are encoded by a single gene, the CALC-I gene. They are expressed in the thyroid and in the nervous system by alternative splicing of the pre-messenger RNA derived from the CALC-I gene. In medullary thyroid carcinoma (MTC), a malignancy derived from the calcitonin-producing C-cells in the thyroid, production of calcitonin and CGRP is a common feature. We investigated the CT and CGRP production of four spontaneous MTCs transplanted three to four times and 14 MTC lines transplanted for several years in WAG/Rij rats, a strain with hereditary MTC. The expression of CT and CGRP in the spontaneous and in the transplanted tumors was studied by means of RNA in situ hybridization (RISH), dot-blot analysis, and immunohistochemistry. A down-regulation of CT production in transplanted compared with spontaneous tumors was observed, but an inverse relation between CT and CGRP mRNA content in both spontaneous and transplanted tumors was not observed. In this study, RISH proved to be as sensitive as dot-blot analysis to detect gene expression in tissue samples. The different approaches of analyzing the gene expression in tissue samples (the cellular localization of gene expression by ISH vs the analysis of an extract of a total tissue sample with dot-blot analysis) showed that each technique is equal in value and that they are complementary to each other.     

Nucleophilic Substitution Reactions of 5-Bromo-6-methyluridines

Abstract

The reactions of the 5-bromo-6-methyl-2′,3′-O-isopropylideneuridines 9 and 10 with a number of nucleophiles in hot DMF have been investigated. With acetate ion as the nucleophile, either the 5-acetoxy- (11,12) or the 6-acetoxymethyl- (15) products can be obtained in modest yield depending upon the exact reaction conditions. With nitrogen nucleophiles (aniline or p-methoxybenzylamine) reaction takes place at the 6-methyl carbon, whereas with sulfur nucleophiles (thiophenol, thioacetate) only the 5-substituted products are obtained.     

Synthesis and Conformational Studies of O 5′, 6-Methanouridine—A New Type of Pyrimidine Cyclonucleoside

Abstract

The 5-oxo-6-methylene-pyrimidine-2,4-dione intermediate (6) that is formed when 5-acetoxy-6-acetoxymethyl-1-β-D-(5-O-acetyl-2,3-O-isopropylidene)-ribofuranosyluracil (5) is treated with sodium hydroxide undergoes cyclization at pH 14 to give 2′,3′-O-isopropylidene-5-hydroxy- O ⁵, 6-methanouridine (8) in good yield. Conversion of 8 into the 5-triflate ester 14 followed by reduction with [(Ph)₃P]₄Pd/Bu₃SnH and deblocking with acetic acid then affords O ^5′, 6-methanouridine (4) Conformational studies (NOE difference spectra, vicinal ¹H-¹³C coupling constants, NOESY and CD spectra, molecular modeling) indicate that the C7-methylene group of 4 projects towards the furanose ring oxygen atom, producing a glycosyl rotation angle of about ? 160°.     

The Conformational Analysis of Substance P Analogs Using High-Field NMR Techniques

Abstract

High-field nuclear magnetic resonance measurements were carried out on substance P fragments SP_4–11 [pGlu⁵]-SP_5–11 and [pGlu⁶]SP_6–11 both at 400 and at 500 MHz. A spectral simulation was carried out on two of these peptides and the coupling constants were interpreted in terms of the conformations. The J_NH-CHa coupling constants are all ～8 Hz, with the exception of glycine, indicating no preferred conformation for the backbone. For the amino acids other than p-Glu, a comparison of the coupling constant data suggests the same relative rotamer populations for the side chains. Proton longitudinal relaxation time data were measured for all three peptides and support the above conclusions.     

Conformational Analysis of the Type II and Type III Collagen α-1 Chain C-Telopeptides by 1H NMR and Circular Dichroism Spectroscopy

Abstract

The type II and type III collagen α-1 chain C-telopeptides are a 27 mer with the sequence NAc- GPGIDMSAFAGLGPREKGPDPLQYMRA and a 22mer, NAc-GGGVASLGAGEKGPVG- YGYEYR, respectively. Their conformations have been studied in CD₃OH/H₂O (80/20) solution by means of two-dimensional proton NMR and CD spectroscopy. Based on TOCSY and NOESY experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H_α coupling constants, sequential and medium range NOEs and amide proton temperature coefficients.

The conformation of the type II C-telopeptide is essentially extended. Evidence from CD spectroscopy suggests that a very minor proportion of the peptide might be helical (ca. 8%), but the NMR data show no evidence for a non-linear structure. The observation of reduced amide proton temperature dependence coefficients in certain sections of the molecule can, in view of the absence of any other supporting evidence, only be interpreted in terms of local shielding from solvent for sterical reasons (large hydrophobic side-chains).

The conformation of the type III C-telopeptide is mostly extended except for a β-turn ranging from Gly⁸ to Glu¹¹, which is stabilized by a hydrogen-bond between NH of Glu¹¹ and the carbonyl group of Gly⁸. The low temperature coefficient of NH(Glu¹¹) and, in particular, the observation of a medium range NOE between H_α (A⁹) and NH(E¹¹) corroborate the existence of a β-turn in this region. Although spectral overlap prevents a precise conclusion with regard to the type of β-turn present, there is some evidence that it might be type II.