Isozymes in nutrient medium of suspension-cultured apple cells (Malus sylvestris Mill.)

The time course of the activities of esterase, -galactosidase, and -glucosidase in cell sap and nutrient medium in in vitro cultured apple cells (Malus sylvestris Mill.) was studied. The corresponding isozyme patterns and the intracellular and extracellular isozyme patterns of acid phosphatase and polyphenol oxidase were compared using isozyme visualization methods adapted to ultra-thin-layer isoelectric focusing. Neither quantitative (total activity) nor qualitative (isozyme pattern) data were congruent for cell saps and nutrient media. Malate dehydrogenase, malic enzyme, and glutamate dehydrogenase occurred in cell sap only. The extracellular activities probably originate to a great part from a programmed release by intact cells. Nutrient media of plant cell cultures constitute a rich source of active plant isozymes.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

“Gomori-positive” neurosecretion in the rat after deafferentation of the medial basal hypothalamus

A portion of the "Gomori-positive" peptidergic neurosecretory (NS) cells in the paraventricular and especially in the supraoptic and postoptic nuclei degenerate three weeks after deafferentation of the medial basal hypothalamus. Most of the remaining NS cells show signs of high activity. Regenerating NS fibres form "muffs" around the blood vessels laterally from the lesion; some of them enter the "isolated" area or persist there if a thin layer of the brain tissue is left somewhere untouched under the basal end of the cut. The regenerating NS fibres are also found outside the nervous tissue: within the scar tissue, in the proliferating connective tissue of the brain sheet below the basal end of the cut and in the mantel plexus area. The NS fibres make close contact with blood vessels invading or penetrating the vascular wall. It is suggested that peptide neurohormones discharged from the "Gomori-positive" NS terminals enter the general blood circulation as well as the portal blood at the site of these newly formed axovasal contacts. It is supposed that under these conditions monoaminergic terminals do not discharge monoamines because no stimulation of monoamine-producing NS cells occurs with deafferentation.     

Photo-CIDNP 1H-NMR of proteins: Exposure,mobility and orientation of the tyrosyl side chains

The photo-CIDNP technique is capable of giving information, not only on the degree of exposure of aromatic residues in proteins and on the correlation time of aromatic side chains, but also on the fine structure of -CH₂ multiplets, whose J-pattern directly reflects the side chain orientation about X ¹. Moreove, the resonance filtering achieved in the difference spectra allows selective measurement of line broadenings in binding processes, which are directly related to the association constants (K _a ). In this communication, data are reported concerning the exposure, mobility and orientation of the tyrosyl side chains in bovine PSTI at different pH values. The immobilization of tyrosyl residues in Met-enkephalin, human, and camel -endorphin in the presence of lipid micelles is also investigated; the extent of interaction of the three peptides with C₁₂PN is discussed. The binding efficiency is considerably lower for the shorter peptide; of the two endorphins, the camel hormone exhibits a higher affinity for the micelles than the human analog.Abbreviations PSTI pancreatic secretory trypsin inhibitor - CIDNP chemically induced dynamic nuclear polarization - C₁₂PN n-dodecylphosphorylcholine - NOE nuclear Overhauser enhancement     

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin (-endotoxin from Bacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac and gna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressing gna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressing cry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.     

Syndecan-4 as a molecule involved in defense mechanisms

Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Syndecan-4-deficient [(Synd4(–/–)] mice were produced to clarify the in vivo role of syndecan-4. Synd4(–/–) mice were more susceptible to -carrageenan-induced nephropathy, and the placental labyrinth from the deficient embryos exhibited more thrombi than wild-type ones. Importantly, Synd4(–/–) mice were more susceptible to endotoxin shock. Further analysis revealed that the mechanism to suppress excessive production of interleukin-1 (1L-1) by transforming growth factor- (TGF-) was impaired in the deficient mice. TGF-, one of the cytokines involved in the suppression mechanism, bound to heparan sulfate chain of syndecan-4, which was induced in macrophages and the microvasculature after administration of lipopolysaccharide. Therefore, augmentation of TGF- function by induced syndecan-4 was suggested as a mechanism of the suppressive action of syndecan-4 against endotoxin shock. Published in 2003.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

The desymmetrisation of endo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrisation adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating an endo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of -turns and parallel -sheets.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

A new approach to predicting protein folding types

A new method is proposed for predicting the folding type of a protein according to its amino acid composition based on the following physical picture: (1) a protein is characterized as a vector of 20-dimensional space, in which its 20 components are defined by the compositions of its 20 amino acids; and (2) the similarity of two proteins is proportional to the mutual projection of their characterized vectors, and hence inversely proportional to the size of their correlation angle. Thus, the prediction is performed by calculating the correlation angles of the vector for the predicted protein with a set of standard vectors representing the norms of four protein folding types (i.e., alla, all ,a+, anda/). In comparison with the existing methods, the new method has the merits of yielding a higher rate of correct prediction, displaying a more intuitive physical picture, and being convenient in application. For instance, in predicting the 64 proteins in the development set based on which the standard vectors are derived, the average accuracy rate is 83.6%, which is higher than that obtained for the same set of proteins by any of the existing methods. The average accuracy predicted for an independent set of 35 proteins of known X-ray structure is 91.4%, which is significantly higher than any of the reported accuracies so far, implying that the new method is of great value in practical application. All of these have demonstrated that the new method as proposed in this paper is characterized by an improved feature in both self-consistency and extrapolating-effectiveness.On sabbatical leave from Department of Physics, Tianjin University, Tianjin, China.     

A cDNA encoding vacuolar type β-d-fructofuranosidase (Osβfruct3) of rice and its expression in Pichia pastoris

A vacuolar type -d-fructofuranosidase (Osfruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type -d-fructofuranosidase of maize (Ivr1). Osfruct3 exists as a single copy per genome. Northern analyses showed that Osfruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Osfruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type -d-fructofuranosidase.     

The two β-oxidation sites in pea cotyledons

Two sites for -oxidation of fatty acids in pea (Pisum sativum L.) cotyledons exist. One site is the microbody, the other the mitochondrion. Mitochondrial -oxidation of fatty acids is carnitine-dependent. The fatty acid permeates the membrane as palmitoylcarnitine which is formed from cytosolic-side palmitoyl-CoA by a carnitine palmitoyltransferase located on the exterior face of the inner mitochondrial membrane as a peripheral protein. A single-gated pore integral membrane translocator is proposed to exchange the palmitoylcarnitine for carnitine or acetylcarnitine across the membrane. An internal (matrix side) carnitine palmitoyltransferase then reforms palmitoyl-CoA which enters -oxidation and subsequently the tricarboxylic-acid cycle.     

The Metallo-β-Lactamases Fall into Two Distinct Phylogenetic Groups

The Ambler Class B metallo--lactamases fall into two distinct phylogenetic groups based on the observation that there is no significant sequence homology between the sequences of members of different groups. Structural alignments confirm that those groups are no more closely related to each other than are the three classes of serine -lactamases, Classes A, C, and D. We present phylogenies of these two groups and suggest a new classification scheme for the -lactamases.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.