Blue guaran (a chromophore-labeled galactomannan) as a reagent for lectins

A new reagent (blue guaran) for quantitative estimation of lectins, has been derived from a galactomannan (guaran). When the lectin solution is added to an aqueous solution of blue guaran, dye-bound guaran is precipitated from the solution. The difference in absorbance of the blue guaran solution before and after the addition of lectin solution is proportional to the amount of lectin present in the sample. The method of preparation of blue guaran, its spectral characteristics and effect of pH on precipitation have also been described. It gives a simple colorimetric method for the estimation of galactose-specific lectins.     

Retention of enzymatic activity by N-terminal domain (1-78) T4-lysozyme: expression of synthetic DNA in Escherichia coli

DNA of 235 b.p. coding for N-terminal domain (1-78) T4-lysozyme was synthesized and cloned by ligating twelve synthetic fragments with a linearized plasmid pUCE8 followed by transformation. On expression in E. coli strain JM103 cells, colonies containing the synthetic DNA were found to be lytic. On purification, clone ptly. 23-5 was found to contain polypeptide (M.W. 10,500), corresponding to N-terminal domain, its dimeric and aggregate form. It was identified by amino acid sequence analysis of the dimeric form.     

Suppression of ouabain-insensitive K-ATPase activity in rabbit nephron segments during chronic hyperkalemia

Recently, we demonstrated that an ATPase stimulated by K (and not inhibited by ouabain, Na-K-ATPase inhibitor) is present in the connecting tubule (CNT) and collecting duct segments of the rabbit. In this study, we determined the effects of high- and low-K diet on K-ATPase activity in the CNT and collecting duct segments of rabbit. One group of animals was given a low-K diet (34 mEq/kg diet) and the other group was given a high-K diet (700 mEq/kg diet) for 1 week. K-ATPase activity was measured by a microfluorometric assay in which ATP hydrolysis is coupled to oxidation of NADH. Low-K animals had plasma K = 3.1 +/- 0.2 as compared with 5.5 +/- 0.5 mEq/l in high-K animals. Low-K animals had significant K-ATPase activity in CNT, CCD (cortical collecting duct) and MCD (medullary collecting duct). On the other hand, K-ATPase activity in all 3 segments from high-K animals was not significantly different from zero. These results support a hypothesis that chronic K loading suppresses the ouabain-insensitive K-ATPase in the distal nephron.     

Immobilization of lipase from Candida cylindraceae and its use in the synthesis of menthol esters by transesterification.

Lipase (EC 3.1.1.3) from Candida cylindraceae has been immobilized by the cellulose-titanium chloride method, and on EP-400 polyethylene, with and without glutaraldehyde crosslinking, to give active preparations when assessed by their ability to catalyse the hydrolysis of tributyrin. In both cases, the use of glutaraldehyde crosslinking was shown to improve the stability of the preparations for repeated use. The lipase immobilized on EP-400 polyethylene was found to be effective in transesterification using tributyrin or triacetin as acyl donors with l-menthol as acceptor. The production of methyl butanoate and of methyl acetate using this immobilized preparation was in each case enhanced in the presence of Amberlite IR 47 Anion exchange resin (OH form).     

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the T1rA and T1rD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related (‘erm-type’) genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.     

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli

Analysis of Tn1725 insertions in the Pif+ plasmid pRS2496 showed the maximum limits of the F pif region to be between 43.7 and 47.15 on the 100-kb map of the F plasmid. The effect of these insertions on the expression of pif polypeptides indicated that two of the pif genes, pifA and pifC, lie within a polycistronic operon.     

Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli.

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.