Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Die Bedeutung der “feedback”-Hemmung der Threonin-Desaminase für die Synthese von Valin und Leucin

Zusammenfassung In Arthrobacter Stamm 23 führte die durch Mutation verursachte allosterische Unempfindlichkeit der Threonin-Desaminase zur dereprimierten Bildung der Enzyme im Isoleucin-Valin-Leucin-Biosyntheseweg. Derepression erfolgte auch, wenn Wildtypzellen in Gegenwart von -Ketobuttersäure inkubiert wurden. In beiden Fällen wurde Isoleucin überproduziert und ins Kulturmedium ausgeschieden. Wie aus Wachstumsexperimenten hervorging, verursachte der Überschuß an -Ketobuttersäure im Medium primär einen Valin- und Leucin-Mangel, der zu einer vorübergehenden Wachstumshemmung führte. Durch die dereprimierte Bildung der Enzyme im Isoleucin-Valin-Biosyntheseweg konnte die Wachstumshemmung überwunden werden.Der vorübergehende Hemmeffekt der -Ketobuttersäure ließ sich auf eine Konkurrenz der Substrate am ersten gemeinsamen Enzym im Isoleucin-Valin-Biosyntheseweg, der Acetohydroxysäure-Synthase, zurückführen. Wegen des niedrigen K _m-Wertes für -Ketobuttersäure wird dieses Substrat vom Enzym bevorzugt umgesetzt. Durch gaschromatographische Bestimmungen der Acetoin- und Acetyläthylcarbinol-Bildung in Enzymtests mit variierten Substrat-Konzentrationen konnte nachgewiesen werden, daß relativ geringe Konzentrationen an -Ketobuttersäure genügen, um die -Acetolacetat-Bildung vollständig zu unterdrücken. Diese Ergebnisse erklären die durch -Ketobuttersäure verursachte vorübergehende Wachstumshemmung bei Bakterien.

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The effect of the feedback inhibition of threonine deaminase on valine-leucine biosynthesis

In Arthrobacter strain 23 the allosteric insensitivity of threonin deaminase caused by mutation resulted in derepressed formation of the enzymes of the isoleucine-valine-leucine pathway. Derepression was also observed, when wild type cells were incubated in the presence of -oxobutyrate. In both cases isoleucine was overproduced and excreted. As growth experiments indicated the excess of -oxobutyrate in the medium caused endogenous valine and leucine deficiency and a transient inhibition of growth. Derepressed formation of the isoleucinevaline biosynthetic enzymes resulted in relief of growth inhibition.The transient inhibitory effect of -oxobutyrate has been traced back to substrate competition at the first enzyme common to the isoleucine and valine pathway, acetohydroxy acid synthase. Due to the low K _m of the enzyme for -oxobutyrate this substrate is preferentially converted. As proven by gaschromatographical measurements of acetoin and acetylethyl carbinol produced in enzyme (acetohydroxy acid synthase) assays with varied substrate concentrations, relatively low concentrations of -oxobutyrate are able to suppress the formation of -acetolactate completely. These results explain the transient inhibitory effect of -oxobutyrate on the growth of bacteria.

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Abkürzungen -KBS -Ketobuttersäure - FAD Flavin-adenin-dinucleotid - AHS Acetohydroxysäure - IPM Isopropylmalat - TPP Thiaminpyrophosphat     

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon γ

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon (IFN) on human Kupffer-cell-mediated cytotoxicity against the SW948 coloncarcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patient who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFN (100 U/ml) or with their combination and the perecentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-(TNF)-sensitive U937 cells as target. The TNF secretion of Kupffer cells was measured and we evaluated the effect of TNF on colon tumour targets. We performed human-Kupffer cell-mediated cytotoxicity blocking experiments with anti-TNF and used paraformaldehydefixed Kupffer cells to demonstrate lysis of TNF-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n=14), and by Kupffer cells activated with hGM-CSF (n=14), IFN (n=6) or their combination (n=6) was respectively: 19.5±2.6%, 25.3±2.9% 41±9.4% and 45.6±8% at E/T=1 and 28.2±2.9%, 35.6±3.2%, 55.6±9.7% and 62.8% at E/T=5. All differences were statistically significant (P<0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNF secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNF had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNF blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNF is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFN in vivo are discussed.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Reversed micelles recognize an active protein

Summary The extraction behavior of native and heated-denatured -chymotrypsin has been investigated with two different reversed micellar systems. A large difference in the degree of extraction was observed for the native relative to the denatured -chymotrypsin. In particular, mixed reversed micelles formulated with DOLPA (dioleyl phosphoric acid) and AOT show a high selectivity for the active -chymotrypsin.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Post-translational proteolytic processing and the isolectins of lentil and other Viciae seed lectins

Electrospray mass spectrometry was used to identify precisely the proteolytic cleavage points within, and at the C-termini of, the proprotein forms of four Viciae lectins that give rise to their two-chain forms. The lectins examined were the pea and lentil lectins, favin and theLathyrus odoratus lectin, which represent each of the four genera in this tribe. The molecular mass data showed single -chain forms for each lectin, with masses consistent with the available sequence and glycopeptide data, indicating that each came from a single proprotein. In contrast, the pea, lentil andL. odoratus -chains occurred in as many as five forms, due to multiple C-terminal cleavage points. Only favin showed a single -chain form. The -chain mass data were again consistent with the sequence information available, except for the lenti lectin -chain which was re-determined by protein sequencing. The two isolectin forms of this protein were shown to arise from -chain species with and without residue Lys53. The mass spectrum of concanavalin A was also examined and both the single-chain form and the two fragment forms showed no evidence of C-terminal heterogeneity.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Acetate inhibition on growth of recombinant E. coli and expression of fusion protein TGFaα-PE40

Summary Acetate was inhibitory to the growth of early induced E. coli cells and their expression of fusion protein, transforming growth factor--Pseudomonas exotoxin 40 (TGF-PE40), but the inhibitory level was strain dependent For E. coli JM109 (pTAC-TGF57-PE40), 2 g/L of added acetate (3 g/L of total acetate in the medium) decreased TGFa-PE40 production by 38.0%. Acetate was less inhibitory to E. coli RR1, and RR1 was not affected by adding 2 g/L of acetate. However, 5 g/L of added acetate (6.7 g/L of total acetate in the medium) decreased TGF-PE40 production by 21.2%. These results indicate that higher acetate concentration was associated with inhibition of TGF-PE40 expression of E. coli JM109 during late induction.     

Syntheses ofγ-fluoro-α-amino acids

Summary Methods for the synthesis of racemic and optically active title compounds are presented. Key step of these four-step procedures is the alkylation with 1-bromo-2-fluoroalkanes of glycine-ester-derived imines in anhydrous medium using lithium diisopropylamide as a base at low temperature or phase transfer catalyzed alkylation with 50% NaOH and triethylbenzylammoniumchloride as the phase transfer catalyst, respectively. Subsequent three-step deprotection gave the free acids in 13–33% overall yield. Deracemization of-fluoro--aminobutyric acid methyl and ethyl esters with-chymotrypsin was shown to give the (–)-enantiomers of the esters and (+)--fluoro--aminobutyric acid in >98% ee, while from thetert-butylester the opposite stereochemical result was observed giving the (–)-acid with 88% ee. Optically active-fluoro--amino acids were synthesized alternatively by phase transfer catalysis with N-benzyl-cinchonium chloride or using an auxiliary-directed asymmetric alkylation of the imine derived from (R)-(+)-camphor or (R)-(+)-2-hydroxypinan-3-one. These processes gave different enantiomers of-fluoro--aminobutyric acid via a monomeric lithium enolate in the first or a dimeric lithium enolate in the second case, respectively. The enantiomeric excess can be improved by lithium/magnesium exchange.     

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like -coixin, the -prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like -coixin genes designated -3A, -3B and -3C were found in the 15 kb -3 genomic clone. The -3A and -3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the -3B gene, suggesting that the three -coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of -coixin clones with the published sequences of 22 kDa -zein and 22 kDa-like -kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like -prolamins and the 19 kDa -zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5 and 3 flanking regions of -3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. –300 prolamin box are present at conserved positions in -3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in -3B that occupies approximately the same positions as those identified for the 22 kDa -zein and -kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like -prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.     

A note on dimensionless life histories for birds versus mammals

Summary Offspring production over the adult lifespan (b/M whereb is the yearly fledgling or offspring production and 1/M is the mean adult lifespan) is an approximate invariant within both birds and mammals. The two taxa differ, however, in that mammals have bothM and b as invariants (b/M = b/M) while birds do not ( is the age at first breeding). Birds have a surprising cancellation in that bothM andb are ^–0.25.     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.     

Lectin analysis of human immunoglobulin G N-glycan sialylation

The lectins Sambucus nigraagglutinin (SNA) and Ricinus communisagglutinin (RCA), specific for 2,6 linked sialylation, and terminal galactose respectively were used to study the occurrence, linkage and distribution of human immunoglobulin G (IgG) sialylation. SNA was shown to bind N-glycan 2,6-linked sialic acid only. Sialidase analysis confirmed that this is the dominant, if not exclusive linkage. Total IgG sialylation was estimated at 1.0[emsp4 ]g SA/mg IgG (or about 0.5 mole per mole) using a biochemical sialic acid assay. SNA displayed strong binding to the IgG Fab fragment in both its native and denatured state. In contrast, SNA failed to bind the IgG Fc fragment in its native form, but displayed strong binding after the Fc was denatured. This allowed the construction of quantitative assays capable of measuring both IgG Fab and Fc 2,6-sialylation without the need for enzymatic peptide digestion.     

Selective inhibition of the non-enzymatic lipid peroxidation of phosphatidylserine in rod outer segments by α-tocopherol

In the present study it was investigated if a-tocopherol shows protection against in vitro lipid peroxidation of phospholipids located in rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe²⁺ system, at 37°C during 160 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of -tocopherol. The fatty acid composition of total lipids isolated from rod outer segment membranes was substantially modified when subjected to non-enzymatic lipid peroxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3). The incorporation of -tocopherol (0.35 mol/mg protein) produce a 43.37% inhibition of the lipid peroxidation process evaluated as chemiluminiscence (total cpm originated in 160 min). The phospholipid species containing the highest amount of docosahexaenoic acid: phosphatidyletanolamine and phosphatidylserine were more affected than phosphatidylcholine during the lipid peroxidation process. Not all phospholipids, however, were equally protected after the addition of -tocopherol to the incubation medium. Phosphatidylcholine and phosphatidyletanolamine, were not protected by -tocopherol, the vitamin provides selective antioxidant protection only for phosphatidylserine. These results indicate that -tocopherol may act as antioxidant protecting rod outer segment membranes from deleterious effect by a selective mechanism that diminishes the loss of docosahexaenoic acid from phosphatidylserine.