Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.     

Lectin binding to cells of Schistosoma mansoni sporocysts and surrounding Biomphalaria glabrata tissue

The binding of different lectins to the surface of mother and daughter sporocysts of Schistosoma mansoni (Trematoda) and to cells of its intermediate host Biomphalaria glabrata (Gastropoda) was investigated. The test system consisted of several biotin-labeled lectins, an avidin-biotin-peroxidase complex and 3,3'-diaminobenzidine. The fixatives used were Formalin, Bouin's and Zenker's solutions; unfixed material was also studied. Most lectins reacted equally with host tissue and parasite tissue. However, receptors for Ulex europaeus I (most probably fucose) were only demonstrated on daughter sporocysts. Thus, a method was found to specifically mark Schistosoma mansoni daughter sporocysts in the digestive gland tissue of its intermediate host. Mother sporocysts and surrounding host tissue differed in their distribution of galactosyl groups. Both lack fucose and N-acetyl-galactosamine. The differences in lectin binding of galactosyl determinants were also observed during the in vitro development of mother to daughter sporocysts.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Origin of ultraviolet damage in DNA

A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.     

Aberrant fatty acyl alpha-hydroxylation in human neuroblastoma tumor gangliosides

Abnormalities of ganglioside structure characterize the neoplastic state, and aberrant glycosylation has been implicated as underlying many new tumor ganglioside structures. However, variations in ceramide structure can also result in novel tumor gangliosides. To address systematically this aspect of ganglioside metabolism, we have initiated a study of the structures of the ceramide species of an oligosaccharide-homogeneous human tumor-derived ganglioside, GM2. The ganglioside was isolated from neuroblastoma tissue and purified by normal-phase high pressure liquid chromatography. Marked ceramide heterogeneity was observed; 18 individual ceramide species of neuroblastoma GM2 were separated by reversed-phase high pressure liquid chromatography and collected. Their structures were determined by a combination of negative- and positive-ion fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry of the underivatized gangliosides. The striking finding was the detection of alpha-hydroxylation of a significant fraction of each of the major fatty acid species (16:0, 18:0, 20:0, 22:0, and 24:1); alpha-hydroxylated species quantitatively represented almost one-fifth of the total tumor GM2 species. Fatty acyl hydroxylation was also detected in the ceramide of several other human tumor gangliosides. In contrast, as previously known, fatty acyl hydroxylation was not detected in the normal human brain gangliosides GM3, GM2, and GM1. We propose that aberrant fatty acid alpha-hydroxylation is a novel and sometimes quantitatively significant characteristic of human tumor ganglioside metabolism.     

A covalently constrained congener of the Saccharomyces cerevisiae tridecapeptide mating pheromone is an agonist

An analog of alpha-factor, the Saccharomyces cerevisiae tridecapeptide mating pheromone (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr), in which the side chains of Lys7 and Gln10 were covalently linked, was synthesized using solid phase methodologies. The yield of the purified cyclic analog cyclo7,10[Nle12]alpha-factor was 30%, and its structure was verified by amino acid analysis, peptide sequencing, fast atom bombardment-mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Cyclo7,10[Nle12]alpha-factor caused growth arrest and morphological alterations in S. cerevisiae MATa cells qualitatively identical to those induced by linear pheromone and was one-fourth to one-twentieth as active as the linear alpha-factor depending upon the S. cerevisiae strain tested. Consistent with the relative activities of the linear and cyclic peptides, binding competition studies indicated that cyclo7,10[Nle12]alpha-factor had approximately 20-40-fold less affinity for the alpha-factor receptor. Hydrolysis of the cyclic peptide by the target cells did not lead to opening of the ring and was less rapid than that of linear alpha-factor. The alpha-factor antagonist des-Trp1-[Ala3,Nle12]alpha-factor reversed the activity of the cyclic analog, and cyclo7,10[Nle12]alpha-factor was not active at the restrictive temperature in a temperature-sensitive receptor mutant. These results support the conclusion that the cyclic alpha-factor occupies the same binding site within the receptor as is occupied by the natural pheromone. The cyclic alpha-factor represents a rare example of an agonist among covalently constrained congeners of small linear peptide messengers.     

Ethanol increases PGE and thromboxane production in mouse pregnant uterine tissue

The teratogenic effect of ethanol in the C57BL/6J mouse can be attenuated by pretreatment with aspirin (ASA). One prominent effect of ASA is to inhibit prostaglandin (PGE) and thromboxane (TXB2) production. We examined the effect of in vivo ethanol exposure on PGE and TXB2 production in a uterine-embryo tissue sample of C57BL/6J mice either before or after in vivo ASA pretreatment on day 10 of gestation. Ethanol increased both PGE and TXB2 production by approximately 20%. ASA caused a marked reduction of PGE and TXB2 in both control and ethanol groups by approximately 80-90%. The mouse strain, gestation time, and study parameters used in this study were the same as in the previously reported ASA attenuation of the teratogenic effect of ethanol. Therefore, the present data add additional support to the hypothesis that prostaglandin and/or thromboxane production may be involved in at least some aspects of fetal alcohol syndrome.