The biosynthesis of cyanogenic glucosides in higher plants. The (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime as intermediates in the biosynthesis of dhurrin in Sorghum bicolor (L.) Moench

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been studied with a microsomal preparation obtained from etiolated seedlings of sorghum. The biosynthetic pathway involves tyrosine, N-hydroxytyrosine, and p-hydroxyphenylacetaldehyde oxime as early intermediates (M?ller, B. L. and Conn, E. E. (1980) J. Biol. Chem. 254, 8575-8583). The use of deuterium-labeled tyrosine and mass spectrometric analyses demonstrate that the alpha-hydrogen atom of tyrosine is retained in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime. This excludes p-hydroxyphenylpyruvic acid oxime as intermediate in the pathway. A high pressure liquid chromatography method was developed to separate the (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime. The microsomal enzyme system was found to produce initially the (E)-isomer of p-hydroxyphenylacetaldehyde oxime. An isomerase then converts the (E)-isomer to the (Z)-isomer, which is the isomer preferentially utilized by the microsomal enzyme system in the subsequent biosynthetic reactions. The (E)-isomer produced in situ is more efficiently converted to the (Z)-isomer than exogenously added (E)-isomer and may thus be metabolically channeled.     

Complete sequences of glucagon-like peptide-1 from human and pig small intestine

In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Estrogen treatment increases phospholipid transfer activities in chicken liver

The effect of subcutaneous beta-estradiol injection on liver phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol transfer activity of immature chicken has been determined. The estrogen administration significantly enhanced the transfer activity of both phosphatidylcholine (100%), phosphatidylethanolamine (160%), and phosphatidylinositol (150%). In vivo experiments revealed that the hormone-induced changes in liver lipid transfer activity were sensitive to a protein synthesis inhibitor, cycloheximide. A partial characterization of liver protein transfer on Sephacryl S-200 showed that multiple transfer proteins are involved in the beta-estradiol effect. This is the first time that hormonal modulation of phospholipid transfer activities is described, and the results suggest that the hepatic phospholipid transfer activities might be involved in the biosynthesis of plasma lipoproteins in vivo.     

Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles

The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points. A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed. By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle. We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA. In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA. The DNA is more accessible in regions near the particle boundary. We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity. Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine. Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle. The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.     

Calcium Activation of Potassium Channels in the Plasmalemma of Eremosphaera viridis

The role of cytoplasmic calcium activity in activation of K⁺-channelsin the unicellular green alga Eremosphaera viridis has beenstudied. As reported previously, after a ‘light off’signal a voltage independent opening of K⁺-channels in the plasmalemmais observed. This effect is indicated by a transient polarization(TP) with a simultaneous increase of the membrane conductance.TPs can also be triggered by different treatments, which allowinvestigations within a ‘short-circuited’ signalchain. (i) After incubation with EGTA a single extended TP canbe released by a sudden increase of the external calcium concentration.The Ca²⁺-channel inhibitors nifedipine (10 ^–2 mol m^–3)and verapamil (5 ? 10^–2 mol m^–3) suppress the releaseof this TP. (ii) In the presence of external calcium the additionof the ionophore A23187 [GenBank] (10–3 mol m^–3) causes anextremely prolonged TP. (iii) Low external concentrations ofbarium (10^–2 mol m^–3) induce repetitive TPs in thepresence of external calcium. In this case the Ca²⁺-channelinhibitors are less effective. (iv) Strontium (0.1–1.0mol m^–3) is able to trigger repetitive TPs even withoutexternal calcium. Whereas barium may stimulate a calcium influx,strontium can serve as a substitute for calcium to induce anopening of K⁺-channels. These results indicate strongly a Ca²⁺-dependentand voltage-independent activation of K⁺-channels in the plasmalemmaof Eremosphaera. The participation of cytoplasmic calcium inthe signal transduction chain after a ‘light off’signal is discussed. Key words: Ca²⁺-dependent K⁺-channels, Ca²⁺-channel effectors, A23187, transient membrane potential, Eremosphaera