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.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Homologous recombination between plasmid DNA molecules in maize protoplasts

Summary The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for -glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB×26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adhl gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 by (Hindlll—PvuII) fragment of the yAdh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3 end of the pB×26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.     

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.     

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.     

Estradiol Protects Against Oxygen and Glucose Deprivation in Rat Hippocampal Organotypic Cultures and Activates Akt and Inactivates GSK-3β

Here we investigated the neuroprotective effect of 17-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3. Our results show a clear neuroprotective effect of 17-estradiol and suggest that this effect could involve PI3-K pathway.     

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.     

Biosynthesis and Molecular Genetics of Clavulanic Acid

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic -lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modfication as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the -lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack -lactamase inhibitory activity.     

A genetic component in human lysosomal enzyme excretion

Acid hydrolases are present in normal human urine in appreciable amounts. Their source appears to be lysosomes released by kidney proximal tubule epithelial cells. For a given lysosomal enzyme the total amount excreted is the product of two parameters, a general one describing the rate of lysosome secretion and a specific one describing the relative concentration of that enzyme in lysosomes. There is considerable population variation in both parameters. Studies of -glucuronidase, -galactosidase, -hexosaminidase, and -galactosidase in monozygotic and dizygotic twins show that an appreciable part of this variation is genetic in origin. This appears to be true for both total enzyme excretion and lysosome composition. Although it was not possible to test directly whether this is also true for the rate of lysosome secretion, the fact that the two former parameters are both heritable strongly suggests that the rate of lysosome excretion is also a heritable trait. Taken together with previous data, the results suggest polygenic control of these biochemical traits. It is particularly significant that -glucuronidase excretion in normal individuals is a heritable trait since the excretion of this enzyme has frequently been used as a measure of normal and pathological physiological changes.This study was supported by grants from the National Institutes of Health (GM-19521) and the Council for Tobacco Research—U.S.A., Inc. (1080). The work was done while the authors were in the Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

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.     

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

Summary A procedure has been developed to isolate protoplasts from mature aleurone layers of the malting variety Alexis and four other barley genotypes. It combines induction of endogenous cell wall degrading enzymes together with use of Onuzuka cellulase R 10 and driselase and results in better yields for two varieties than can be obtained with the huskless variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early developmental stage, as judged by ultrastructure. Gibberellic acid-induced changes in protoplast structure resemble those reported for Himalaya protoplasts. The protoplasts secrete both -amylase (EC 3.2.1.1) and (1-3, 1-4)--glucanase (EC 3.2.1.73) into the surrounding medium. Transfection studies using a low pI -amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts from Alexis and Himalaya revealed significant differences in their hormone responsiveness. In the absence of hormones, low levels of expression of the reporter enzyme were obtained in Alexis protoplasts, while high levels were characteristic for Himalaya protoplasts. An 8-fold increase in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A₃, whereas expression in Himalaya protoplasts remained unchanged. When Himalaya protoplasts were isolated from aleurone layers that had not been incubated with GA₃ during the initial stages of protoplasting (the classical procedure), the hormone response of the promoter was 2.5-fold. It is thus possible to optimize the aleurone protoplast isolation procedure for different barley genotypes and mutants of interest in studies of transgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.Abbreviations ABA abscisic acid - APIM aleurone protoplast isolation medium - CAT chloramphenicol acetyltransferase - EDTA ethylenediamine tetraacetic acid - ER endoplasmic reticulum - GA₃ gibberellin A₃ - IgG immunoglobulin G - MES 2-(N-morpholino)-ethanesulfonic acid - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - pI isoelectric point - PIPES piperazine N,N-bis-(diethanesulfonic acid) - SDS sodium dodecyl sulfate     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis

Summary The expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron-microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of -type cytokeratins in the -keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with -and -type keratin filament bundles in the -keratin-rich layers of epidermis known as the beta stratum.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Isolation and Properties of Major Components of Penicillium canescens Extracellular Enzyme Complex

The composition of the enzyme complex secreted by Penicillium canescens was investigated. A scheme for purification of the main components of the complex by chromatofocusing on a Mono P column was developed. It was found that along with beta-galactosidase, the major components of the complex were endo-beta-1,4-xylanase (31 kD, pI 8.2-9.3), alpha-L-arabinofuranosidase (60 kD, pI 7.6), arabinoxylan-arabinofuranohydrolase (70 kD, pI 3.8-4.0), and endo-beta-1,3/1,4-glucanase (40 kD, pI 4.4). The substrate specificity, pH and temperature activity optima, adsorbability, thermal stability, and ability for synergic interaction of the isolated enzymes were studied.     

Enzymic synthesis and hydrolytic resolution of alkyl β-d-glucopyranosides

Extracellular -glucosidases from Aspergillus oryzae (two strains), Fusarium oxysporum, Aspergillus terreus and Talaromyces flavus were either induced by cellobiose or 6-deoxyglucose, or produced without induction, i.e., either on casein acid hydrolyzate or on the Sabouraud medium. They were tested to catalyze a stereoselective synthesis of the cis- and trans-isomers of 2-(4-methoxybenzyl)-1-cyclohexyl -d-glucopyranosides with either d-glucose or phenyl -d-glucopyranoside as sources of the glucosyl unit. The enzymes also hydrolyzed (resolved) diastereoisomeric mixtures of alkyl -d-glucopyranosides, which were prepared by the Koenigs–Knorr method from the respective cis- and trans-isomers of 2-(4-methoxybenzyl)-1-cyclohexanol.     

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.     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.