The covalent structure of cartilage collagen. Amino acid sequence of residues 552–661 of bovine α1(II) chains

The covalent structure of the first 111 residues from the N-terminus of peptide α1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552–661 of the α1(II) chain. The sequence was determined by automated Edman degradation of peptide α1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the α1(II) chain with the homologous segment of the α1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the α1(II) chain (Butler, Miller & Finch (1976) Biochemistry 15, 3000–3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the α1(II) chain were compared with the homologous regions of the α1(I) and α2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the α1(I) chain, it appears that the α1(II)-chain sequence presented here contains three more amino acids than that reported for the α1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide α1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

High resolution 2-deoxyglucose localization in olfactory epithelium

The olfactory epithelium of the salamander and the mouse hasbeen analyzed for patterns of activity elicited by odor stimulation.A high-resolution adaptation of the 2-deoxyglucose (2DG) methodwas used, involving freeze-substitution in anhydrous acetonecombined with nuclear emulsion autoradiography, according toSejnowski et al. (1980). In animals exposed to the odor of amylacetate, the autoradiograms of 2 µg thick sections showedrestricted regions of high [¹⁴C]2DG and [³H]2DG uptake. Withinthese regions, there was a characteristic pattern of diffuselocalization in the superficial supranuclear zone; small clumpsor strands of grains in the receptor cell nuclei layer; andthick clumps of grains in the basal cell layer. The relationof these patterns of differential odorinduced activity in thecells of the epithelium is discussed. ^* Present address: Department of Membrane Research, The WeizmannInstitute of Science, Rehovot, Israel     

Determination of 2,4-diamino-5-benzylpyrimidines in combination with sulphadiazine in biological fluids using high-performance liquid chromatography

High-performance liquid chromatographic methods for the simultaneous analysis of tetroxoprim (TXP)/sulphadiazine (SDZ) and metioprim (MTP)/SDZ in serum and prostatic secretion (PS) are described. The detection limits in serum and PS were 50 ng TXP per ml and 100 ng SDZ per ml, and 40 ng MTP per ml and 100 ng SDZ per ml, respectively. The intra-assay coefficients of variation were in the range of 2.7–2.19%. Some preliminary data from a pharmacokinetic study in geriatric patients and a distribution study in dogs are presented. These methods enable the investigator to process a large number of TXP/SDZ and MTP/SDZ samples in one working day.     

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.     

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Receptor-independent catabolism of low density lipoprotein. Involvement of the reticuloendothelial system

In this study we examine the effects of intravenous ethyl oleate emulsions on the metabolism of native and cyclohexanedione-modified human low density lipoprotein in rabbits. Treatment produced a highly significant fall in receptor-independent catabolism as measured by the fractional clearance rate of cyclohexanedione-modified low density lipoprotein. Receptor-dependent catabolism (the difference between the fractional clearance rates of native and cyclohexanedione-modified low density lipoprotein) was variably affected with some animals showing a decrease in receptor activity. These data suggest that the reticuloendothelial system makes a substantial contribution to receptor-independent low density lipoprotein catabolism in the rabbit.