Radioiodination of proteins using prosthetic group: a convenient way to produce labelled proteins with in vivo stability.

Radiolabelled peptides can provide new approaches for radiopharmaceutical development. Several prosthetic groups have been developed for radioiodination of proteins in order to minimize in vivo dehalogenation. In this work, the prosthetic group N-succinimidyl 4-[131I]iodobenzoate ([131I]SIB) was obtained by an alternative procedure that employs Cu(I) assisted radioiododebromination to produce p-[131I]iodobenzoic acid with a radiochemical yield of 92.73 +/- 1.51% (N = 6), followed by the reaction with TSTU (O-(N-succinimidyl)-N,N,N'N'-tetramethyluronium) in alkaline medium. The HPLC profile of the final product, revealed that [131I]SIB was obtained with a radiochemical purity of 98.19 +/- 1.14% (N = 6 Swiss mices (normal group) and animals with inflammation focus developed on the right thigh by tupertine injection) were injected with human immunoglobulin (IgG) radioiodinated with [131I]SIB and by direct method (Iodogen). The comparison of results showed a fast blood clearance, better target organ/background relation and low uptake in thyroid and stomach (p < 0.01) for the protein labelled with [131I]SIB, what suggests a greater in vivo stability.     

Effect of lectins on the transport of food ingredients in Caco-2 cell cultures

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03 approximately 2 mmol/L) in the presence or absence of lectins (1 approximately 180 microg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway.     

Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.     

Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.     

A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant

Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses.     

High-level expression and characterization of fully active recombinant conger eel galectins in Eschericia coli

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Indolepyruvate Pathway for Indole-3-Acetic Acid Biosynthesis in Bradyrhizobium elkanii

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatantof Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp)was incorporated into IAAId and indole-3-acetic acid (IAA),suggesting that B. elkanii produces IAA via IAAId from Trp.In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA)was converted to IAAId, and exogenously added IAAId was rapidlyconverted to IAA. Furthermore, the activity of indolepyruvatedecarboxylase (IPDC), which catalyzes the decarboxylation ofIPyA to produce IAAId and is a key enzyme for IPyA pathway,was detected in B. elkanii cell-free extract. The IPDC activitydepended on Mg²⁺ and thiamine pyrophosphate, cofactors of decarboxylation.This mounting evidence strongly suggests that IAA synthesisoccurs via IPyA pathway (Trp IPyA p IAAId IAA) in B. elkanii. (Received December 11, 1995; Accepted March 4, 1996)