Exposure of tryptophanyl residues inα-lactalbumin and lysozyme

Summary The effect of iodide ion on the tryptophyl fluorescence of the homologous proteins lysozyme and-lactalbumin in their native form, as well as in their modified structures and in fragments from these proteins was studied. By assessing the contribution to the total fluorescence of the exposed and buried Trp residues, and of the respective fluorescence quantum yields, the quantization of the number of Trp exposed to the solvent for all the species studied was possible. Both native proteins show an important increase in the number of Trp residues exposed to the solvent when treated with denaturing agents. The peptides L-II (aa 13-105) and-I (aa 1-90) from lysozyme and-lactalbumin, respectively, showed Trp residues with different degree of exposure, whereas the smaller fragments, L-III (aa 106-129) and-II (aa 91–123), had all their Trp residues exposed to the solvent.     

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.     

Molecular recognition between 4a<Emphasis Type="Italic">S/R</Emphasis>-galanthamine diastereoisomers and α-cyclodextrin

Molecular recognition between 4aS/R-galanthamine diastereoisomers (1: 4aS-galanthamine; 2: 4aR-galanthamine) and -cyclodextrin (-CD) were studied by use of docking and molecular dynamics (MD) simulation approaches. The binding energy of constructed 2···-CD complexes is ~17 kcal mol^–1 lower than that of 1···-CD, implying a stronger binding ability of 2 with -CD than that of 1. The theoretical modeling result is consistent with our previous CZE result, which demonstrated that -CD is an efficient chiral additive for separating 1 and 2. The modeling result also indicates that both hydrophobic interaction and H-bond force may work as major factors for molecular recognition between the galanthamine diastereoisomers and -CD. Figure Chemical structures of 4aS-galanthamine (left) and 4aR-galanthamine (right)Abbreviations Galanthamine 4aS,6R,8aS-4a,5,9,10,11,12-Hexahydroxy-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-e,f]benzazepin-6-ol     

Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. These processing enzymes belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. We isolated two genes of VPEs from a genomic library of Arabidopsis. The gene products were designated -VPE and -VPE, and they were 56% identical in terms of amino acid sequence. The amino acid sequences of -VPE and -VPE were also 55% and 67% identical to that of castor bean VPE, respectively. The gene for -VPE had 7 introns, while that of -VPE had 8 introns. Northern blot analysis revealed that -VPE is expressed in rosette leaves, cauline leaves and stems of Arabidopsis, while -VPE is predominantly expressed in the flowers and buds. Neither -VPE nor -VPE is expressed in the siliques. This result strongly suggests that the isolated genes encode isozymes of VPE that are specific to vegetative organs.     

Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment

Novel NMR pulse schemes for simultaneous measurement of ¹ D _CHand ² D _NHresidual dipolar couplings in proteins is presented. We show that ² D _NHcoupling can be very useful for protein structure determination. The ² D _NHcoupling can be measured from ¹⁵N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large ¹ D _CHcoupling. The dynamic range of ² D _NHcoupling is comparable to ¹ D _NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on ¹⁵N/¹³C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both ¹ D _CHand ² D _NHcouplings, was obtained.     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

α-Amylase structural genes in rye

Summary Rye -Amy1, -Amy2, and -Amy3 genes were studied in the cross between inbred lines using wheat -amylase cDNA probes. The -Amy1 and -Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the -Amy3 region was much more conserved. The numbers of restriction fragments found and the F₂ segregation data suggest that there are three -Amy1 genes, two or three -Amy2 genes, and three -Amy3 genes in rye. These conclusions were supported by a simultaneous study of -amylase isozyme polymorphism. The F₂ data showed the three individual -Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two -Amy2 genes were shown to be separated by 5 cM. Linkage data within -Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Autosomal dominant polycystic kidney disease and α−4.2 thalassemia in a Caucasian family

Summary We describe the first known association between autosomal dominant polycystic kidney disease (ADPKD) and ^–4.2 thalassemia in a Caucasian family. Linkage studies have been carried out using two probes (3 HVR and 24-1) linked to ADPKD on locus PKD1 and two probes (1-PstI and BarnH-I/EcoRI-2 fragment) allowing detection of -thalassemia with either a 3.7-kb deletion or a 4.2-kb deletion. Our results show that to avoid misinterpretation it is important to investigate the occurrence of an -gene deletion when polymorphisms situated in the -globin locus are used for linkage studies on ADPKD. The studied family is one of the rare cases of leftward deletional thalassemia described in a non-Asian population.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Ontogeny of estrogen receptor (ER) alpha and its co-localization with pituitary hormones in the pituitary gland of chick embryos

Estrogen is involved in regulating the development and hormone secretion of the anterior pituitary gland following its binding to estrogen receptors (ERs) expressed on pituitary cells. However, the pituitary is comprised of several cell types, and to date, there is no data about the specific cell types expressing ERs in embyonic chick pituitary. We therefore followed, by immunohistochemistry, the ontogeny of the pituitary ER alpha (ER), and the cell types expressing ER throughout chick embryo development. ER immunoreacitivity was restricted to the nuclei of pituitary cells. ER-immunopositive (ER⁺) cells were first detected at embryonic day 6.5 (E6.5), after which ER⁺ cells were consistently detected throughout the anterior pituitary gland, although the density of ER⁺ cells in the caudal lobe of the pars distalis was higher than that in the cephalic lobe. The proportion of ER⁺ cells in the pituitary was about 6% at E8.5; expression increased to 22% by E18.5 of gestation, with no additional change until hatching. Double-labeling of ER and pituitary hormones showed that the dominant cell types expressing ER were gonadotrophs immunopositive for luteinizing hormone (LH); the proportion of ER⁺ cells expressing LH increased throughout gestation and reached approximately 57% at hatching. About 2%–6% of thyroid-stimulating-hormone-immunopositive and 1%–2% prolactin-immunopositive cells expressed ER at later stages of embryonic development, but no growth-hormone-positive or adrenocorticotropic-hormone-positive cells expressed ER during the embryonic period. Thus, gonadotrophs are the main cell population expressing ER in the anterior pituitary gland of chick embryo, and ER is involved in regulating the development of the pituitary gland and the maturation of the hormone-secreting function.This work was supported by grants from the Natural Science Foundation for Outstanding Young Scientists of China (30325034) and the Natural Science Foundation of China (30170693, 30471264).     

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-α-cyclodextrin complex

Newborn rat adrenocortical cells were successfully cultured in a serum free carrier protein free medium (SPFM) by using -cyclodextrin as a cholesterol carrier and have expressed corticosteroid biosynthesis in this medium. A stable inclusion complex of cholesterol--cyclodextrin with a molar ratio of almost 1 was obtained for a 5 × 10^–5 mol/1 -cyclodextrin concentration. Cell cultures incubated with [4-¹⁴C] cholesterol--cyclodextrin in SPFM produced, under ACTH stimulation, various ¹⁴C labeled steroids with a predominance of corticosterone and 18-hydroxy-11-deoxycorticosterone. As measured by gas chromatography and mass spectrometry, the ratio between corticosteroids (21-hydroxylated steroids) and 20-reduced steroids produced in SPFM with cholesterol--cyclodextrin was equal to 1.8. This corresponds to a value of 3.6 times higher than that found in the serum free medium with cholesterol-albumin. Consequently, the chemically defined SPFM with cholesterol--cyclodextrin used in this study is more suitable for corticosteroidogenesis by adrenal cells in culture than a serum free medium with cholesterol-albumin.Abbreviations -CD -cyclodextrin - ACTH adrenocorticotropic hormone - 20-dihydroprogesterone 20-hydroxy-4-pregnene-3-one - 11-hydroxy-20-dihydroprogesterone 11, 20-dihydroxy-4-pregnene-3-one - 11-hydroxyprogesterone 11-hydroxy-4-pregnene-3,20-dione - C--CD cholesterol--cyclodextrin complex - corticosterone 11,21-dihydroxy-4-pregnene-3,20-dione - deoxycorticosterone 21-hydroxy-4-pregnene-3,20-dione - 18-hydroxy-11-deoxycorticosterone 18,21-dihydroxy-4-pregnene-3,20-dione - 18-hydroxy-20-dihydroprogesterone 18-20-dihydroxy-4-pregnene-3-one - 18-hydroxyprogesterone 18-hydroxy4-pregnene-3,20-dione - progesterone 4-pregnene-3,20-dione - SFM-S serum-free medium - SPFM serum-free protein-free medium - SSM serum supplemented medium     

α-Globin gene deletions associated with αAand αG Philadelphiagenes in an Algerian family that includes two Hb G homozygotes

Summary An Algerian family with a high degree of consanguinity and including two homozygotes for Hb-G Philadelphia is presented. Whether homozygotes or heterozygotes, all subjects displayed microcytosis (with various degrees of poikilocytosis) and a moderately depressed -globin chain synthesis. Hb H and Heinz bodies were absent. DNA mapping revealed the presence of 3.7 kb deletion resulting from the rightward type of recombination event between ₂ and ₁ genes on both the ^A/and the ^G chromosomes. Such data indicate that the –^A/ and –^G/ haplotypes are involved and suggest that the –^G/ haplotype, which is very rare in Algeria, has an African Black origin. In subjects with genotype (–^A/–^G/) or (–^G/–^G/), the output of the remaining genes is sufficiently high to avoid the appearance of Hb H. This situation contrasts with that reported in an Algerian patient, who had a (–^A/–^A/) genotype but who was producing Hb H (Whitelaw et al. 1980). The data collected from this family suggest that the –^A/ haplotypes are heterogeneous in Algerians.     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

Generation and characterization of an HLA-DRα-specific monoclonal antibody using L-cell transfectants expressing human and mouse class II major histocompatibility dimers

Among the HLA-DR-specific monoclonal antibodies (mAbs) that have been characterized previously, there is a marked shortage of DR chain-specific mAbs which stain unfixed cells. This may result from the high degree of sequence similarity between the ₁ domains of HLA-DR and H-2E leading to a state of cross-tolerance to DR in H-2-expressing mice. BALB/b (E-negative) mice were immunized with DR1-transfected mouse L cells. The chain specificity of the resulting DR-specific mAb was determined using a panel of transfectants expressing hybrid mouse/human class II heterodimers. A DR-specific mAb was generated which was capable of immunoprecipitating DR dimers and inhibiting the anti-DR alloresponse of human T-cell clones. The present study demonstrates that, with the selection of suitable recipient strain, transfectants can be useful in the generation and definition of chain-specific mouse mAbs.     

Studies of the mechanisms of toxicity of the administration of recombinant tumor necrosis factor α in normal and tumor-bearing mice

Summary Tumor-bearing mice have a greater sensitivity to the acute lethal effects of the administration of high-dose recombinant human tumor necrosis factor (rhTNF-) compared to normal, non-tumor-bearing mice. We studied whether or not the presence of tumor per se was responsible for the enhanced rhTNF- toxicity. Tumor-bearing mice underwent tumor excision or sham operation before the systemic administration of rhTNF at staged times (0.5–24 h) following surgery. There was little survival difference between sham-operated tumor-bearing mice and tumor-bearing mice undergoing tumor excision (at 24 h, treatment with 12 µg rhTNF-, survival:sham-operated tumor bearers = 0/12, excised tumor-bearers = 0/12;P ² <0.01 compared to non-tumor-bearers). Mice without tumors receiving sham operation, had minimal toxicity (10 of 12 mice surviving). The injection of 3 ml Ringer's lactate i.p. before i.v. rhTNF- therapy increased survival in tumor-bearing animals; following pretreatment with Ringer's lactate 30/42 mice survived 12 µg rhTNF- compared to 6/42 surviving a similar rhTNF- dose without hydration (P ² <0.001). Since the production of oxygen free-radical metabolites has been postulated to play a role in the acute toxicity of rhTNF-, bismuth subnitrate was used to induce the enzyme metallothionein to act as a natural scavenger for these metabolites. Daily oral bismuth subnitrate treatments improved survival of mice with MCA-106 or MCA-102 sarcoma and of mice without tumors, with higher rhTNF- doses (12–20 µg), without reducing the therapeutic effect of rhTNF- against the weakly immunogenic MCA-106 sarcoma. These studies suggest methods for reducing the toxicity of rhTNF- administration in clinical trials.     

Selective inhibition of transition from sexual agglutination to zygote formation by ethyl N-phenylcarbamate in Saccharomyces cerevisiae

Effects of ethyl N-phenylcarbamate (EPC) on the mating reaction of Saccharomyces cerevisiae were studied, with special attention on the effect on the pheromone action. EPC inhibited zygote formation at a concentration which promoted induction of sexual agglutinability. EPC enhanced agglutinability induction by pheromone, but inhibited -pheromone-induced formation of large pearshaped cells in a mating type. The enhancement of agglutinability induction was accompanied with increased production of a agglutination substance and inhibition of pheromone inactivation. EPC arrested the cell cycle of a cells probably in the step controlled by CDC19, CDC35, cAMP etc., just before the step controlled by CDC28, pheromone etc.Abbreviations EPC Ethyl N-phenylcarbamate - PBS 0.01 M phosphate buffer solution, pH 5.5 - SPB spindle pole body     

Facile preparation of optically pure [α-2H]-α-amino acids

Summary Racemic [-²H]--amino acids were prepared by heating the corresponding amino acids (Phe, nor-Leu and Dopa) with 0.05 equivalents of benzaldehyde in deuterated-acetic acid. Based on¹H-nmr measurement, the isotopic purities of these racemized [-²H]--amino acids were found to be higher than 99.5%. Methylation of these isotope-labelled amino acids was achieved in methanol/thionyl chloride without affecting isotopic purity. Optically pure [-²H]--amino acids were obtained in high yield with high enantiomeric excess via alcalase catalysed resolution.