Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Conformational stability of α-lactalbumin missing a peptide bond between Asp66 and Pro67

The peptide bond between Asp⁶⁶-Pro67 of -lactalbumin was cleaved with formic acid (cleaved-lactalbumin). Secondary structural changes of the cleaved-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleaved-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism spectrum. The cleaved-lactalbumin contained 29%-helical structure as against 34% for the intact protein. Some helices of the cleaved-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4mM SDS. On the other hand, the helical structures of the cleaved-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no diffrence was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65°C and recovered upon cooling.     

Compact state of a protein molecule with pronounced small-scale mobility: bovine α-lactalbumin

We describe a novel physical state of a protein molecule which is nearly as compact as the native state and has pronounced secondary structure, but differs from the native state by the large increase of thermal fluctuations (in particular, by the large mobility of side groups). This state has been characterized in detail for the acid form of bovine -lactalbumin as a result of the study of physical properties of this state by a large variety of different methods (hydrodynamics, diffuse X-ray scattering, circular dichroism and infrared spectra, polarization of the luminescence, proton magnetic resonance, deuterium exchange and microcalorimetry). It has been shown that bovine -lactalbumin can be transformed into a similar state by thermal denaturation. This process is thermodynamically two state (i.e. all-or-none transition), which means that this state differs from the native one by a phase transition of the first order.Abbreviations B-LA bovine -lactalbumin - Gu·HCl guanidine hydrochloride - CD circular dichroism - UV ultraviolet - IR infra-red - NMR nuclear magnetic resonance. Differen forms of B-LA are abbreviated as follows - N native form - A acid form - T temperature-denatured form - U unfolded form (by 6 M Gu·HCl or 8 M urea). All forms have intact S-S bonds     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased     

The effect of modification and fragmentation of α-lactalbumin on lactose and lactosamine synthase reactions

In the presence of the modifier protein -lactalbumin, bovine milk galactosyltransferase transfers galactose to glucose forming lactose instead of transferring toN-acetylglucosamine formingN-acetyllactosamine. At low concentrations of -lact-albumin, the lactosamine synthase activity is stimulated by -lactalbumin and decreases when the lactose synthase activity develops along a sigmoidal curve. The observation suggests that different interactions between -lactalbumin and enzyme were responsible for the modulating effect of the -lactalbumin in the lactose and lactosamine synthase reactions.To study the nature of the protein-protein interactions, -lactalbumin was both modified and cleaved chemically. Reduction and alkylation with iodoacetic acid, iodoacetamide or 4-vinylpyridine abolished the ability of the -lactalbumin to induce lactose synthase activity but stimulated lactosamine synthase activity 7-to 12-fold.A peptide fragment corresponding to residues 26–60 of -lactalbumin isolated from a 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene (BNPS-skatole) fragmentation of the molecule was active in the lactosamine but not lactose synthase reaction. We concluded that, whereas lactose synthase required -lactalbumin, in the native conformation, lactosamine synthase activity was stimulated by a linear sequence of amino acids in peptide 26–60.Abbreviations MES 4-N-morpholinoethanesulfonic acid - TRIS 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP-Gal uridinediphosphogalactose - BNPS-skatole 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene - EDTA ethylene diamine tetra acetic acid     

Conformational changes of α-lactalbumin and its fragment, Phe31-Ile59, induced by sodium dodecyl sulfate

Conformational changes of bovine -lactalbumin in sodium dodecyl sulfate (SDS) solution were studied with the circular dichroism (CD) method using a dilute phosphate buffer ofpH 7.0 and ionic strength 0.014. The proportions of -helix and -structure in -lactalbumin were 34% and 12%, respectively, in the absence of SDS. In the SDS solution, the helicity increased to 44%, while the -structure disappeared. In order to verify the structural change from -structure to -helix, the moiety, assuming the -structure in the -lactalbumin, was isolated by a chymotryptic digestion. The structure of this -lactalbumin fragment, Phe³¹-Ile⁵⁹, was almost disordered. However, the fragment adopted a considerable amount of -helical structure in the SDS solution. On the other hand, the tertiary structure of -lactalbumin, detected by changes of CD in the near-ultraviolet region, began to be disrupted before the secondary structural change in the surfactant solution. Dodecyl sulfate ions of 80 mol were cooperatively bound to -lactalbumin. Although the removal of the bound dodecyl sulfate ions was tried by the dialysis against the phosphate buffer for 5 days, 4 mol dodecyl sulfates remained per mole of the protein. The remaining amount agreed with the number of stoichiometric binding site, determined by the Scatchard plot, indicating that the stoichiometric binding was so tight.     

Synthesis of New 15-Ketosterol Analogues from Ergosterol

Ergosteryl acetate was converted through three stages into 3-acetoxy-24-methyl-5-cholesta-8(14),22-diene-15-one in 32% overall yield. The product was transformed to 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, and 24-methyl-5-cholesta-8(14),22-diene-3,15-dione. The compounds were characterized by ¹H and ¹³C NMR spectra.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Identification of human milk α-lactalbumin as a cell growth inhibitor

Summary A growth inhibitory protein, mammary inhibitory activity (MIA), was purified to apparent homogeneity from human milk. At concentrations of 5 to 10 ng/ml, the factor inhibited the growth of mammary epithelial cells by 30–80% and also inhibited the growth of normal rat kidney cells. Whereas the cell division of normal human mammary epithelium in primary culture was inhibited by MIA, cell division by fibroblasts from the same tissues was unresponsive. Inhibition was dose and time dependent and readily reversed when MIA was removed. MIA also inhibited growth in culture for three cell lines. The growth inhibitory protein migrated as a 14 kDa protein under reducing conditions on polyacrylamide gels in the presence of sodium dodecyl sulfate. The apparent isoelectric point was pI 5.0. The amino acid composition of MIA resembled that of -lactalbumin, and sequence analysis of the N-terminal region comprising residues 1–24 and an isolated peptide were identical with the N-terminal and residues 66–81 of human -lactalbumin. In addition, MIA was active in the lactose synthase system. The results strongly suggest that MIA and -lactalbumin are identical proteins. Consistent with these results, -lactalbumin preparations from several mammalian species, including human, goat, cow and camel, were all found to be growth inhibitory for cultured mammary epithelial cells. The inhibitory activity associated with human -lactalbumin was destroyed by digestion with pepsin or chymotrypsin, by carboxymethylation of cysteine, or by cleavage of methionine 90 following cyanogen bromide treatment. The results raise the possibility that during lactation -lactalbumin, a product of mammary cell differentiation, could be a physiologically relevant feed-back inhibitor of mammary cell growth and perhaps of other cell types as well.Abbreviations MIA mammary inhibitory activity - MDGI mammary derived growth inhibitor - -LA alpha lactalbumin - H--LA human -lactalbumin - NRK normal rat kidney - IMEM improved minimal essential medium - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - EGF epidermal growth factor - TGF transforming growth factor - CNBr cyanogen bromide - SDS sodium dodecyl sulfate - kDa kilodaltons - ND-PAGE non-denaturing polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

α-Globin gene deletion causes α-thalassemia syndromes in two German families

Summary Restriction endonuclease mapping of chromosomal DNA has been used to determine whether the -globin gene deletion or non-deletion form of -thalassemia is the underlying molecular defect in individuals of two unrelated German families with -thalassemia syndromes. The obtained DNA pattern in all cases indicated loss of -globin genes resulting in-/,--/, and--/- genotypes in thalassemia-2, -thalassemia-1, and Hb H individuals respectively. The chromosomes showing loss of one -globin gene in -thalassemia-2 and Hb H disease were characterized by the so-called rightward deletion form exhibiting loss of a 3.7 kb DNA fragment in the -gene cluster.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

Oxidation of 17α,20β- and 17α,20α-Dihydroxypregn-4-en-3-ones,Side Products of Progesterone Biotransformation with Recombinant Microorganisms Expressing Cytochrome P-45017α

Progesterone biotransformation with recombinant yeasts Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 expressing bovine adrenocortical cytochrome P-45017 yielded 17-hydroxyprogesterone and two diols, 17,20- and 17,20-dihydroxypregn-4-en-3-ones. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17–20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17-hydroxylation and 20- and 20-reduction. The results widen the possibilities of enzymatic and chemical modifications of steroids.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Fractionation of whey proteins with a hexapeptide ligand affinity resin

The isolation of individual proteins from whey would allow production of more consistent and reliable products by the food industry and possibly would also increase their use in the pharmaceutical industry. -Lactalbumin is the second most prevalent protein in bovine milk whey and has many uses including serving as an excellent protein source in infant formulas, power drinks and other beverages that require soluble, nutritional protein. In this study, we describe two methods for production of -lactalbumin from whey protein isolate using bioselective adsorption. The use of a peptide ligand (WHWRKR) attached to a resin allowed production of an -lactalbumin-rich fraction with a purity of 90.6% and a recovery of 47.9%, while also producing other fractions of commercial interest. The combined use of an amino resin followed by the WHWRKR resin produce a highly purified -lactalbumin (100%) with a yield of 35.2%.