The entry process as the target for energy input in active transport of α-aminoisobutyric acid byMycobacterium phlei

Mycobacterium phlei was shown to accumulate -aminoisobutyric acid, establishing a concentration gradient of approximately 15,000-fold. The apparent affinity constant of the carrier for -aminoisobutyric acid was 1.8 µM. The system exhibited a broad specificity provided two structural requirements were satisfied: the presence of a free amino and carboxyl group on the alpha carbon and the absence of a net charge. The role of energy coupling on the accumulation of -aminoisobutyric acid was studied by two different kinds of experiments, the relative effects of the inhibitors on the rate of entry and the steady-state of accumulation, and a comparison of the efflux induced at the final steady state by the addition of (a) excess nonradioactive -aminoisobutyric acid, (b) energy inhibitors, or (c) both. The results are consistent with the hypothesis that accumulation of -aminoisobutyric acid is due to an increased rate of entry, the rate of exit not being affected by metabolic inhibitors.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Uptake and incorporation of pyrimidines in Euglena gracilis

In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concentrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance -AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and -AIB is inhibited by cycloheximide in a different manner after 5–10 min.Abbreviations Barb barbituric acid - 5-BrU 5-bromouracil - Cyd cytidine - Cyt cytosine - DHU dihydrouracil - dUrd deoxyuridine - dThd thymidine - 5-FU 5-fluorouracil - Ora orotic acid - Thy thymidine - Ura uracil - Urd uridine - CHI cycloheximide - -AIB -aminoisobutyric acid Dedicated to Prof. Dr. Dr. h.c. mult. K. Mothes on the occasion of his 75th birthday     

A retroposon-like short repetitive DNA element in the genome of the human blood fluke,Schistosoma mansoni

The genome of Schistosoma mansoni, a human blood fluke, contains a family of short repetitive DNA elements which we have named the SM family. In this paper we report the sequences of two SM family members which are derived from tandem arrangements and four family members which are dispersed copies. The two tandemly repeated copies are 331 and 335 bp, while the four dispersed copies range in size from 107 to 322 bp. Three dispersed copies are flanked by direct repeats and have AT-rich 3 ends. The tandem copies and one of the dispersed copies have regions of homology to RNA polymerase III promoters and arginine tRNA genes. In addition the repeated element is rearranged in two of the dispersed copies when compared with the other dispersed and two tandem copies. Localization studies show that SM elements are distributed in the sex and autosomal chromosomes. These observations suggest that members of this family may have been dispersed throughout the genome via RNA intermediates.     

Endotoxin-induced release of tumour necrosis factor and interferon in vivo is inhibited by prior adrenoceptor blockade

Summary The effect of - and -adrenoceptor blocking agents on endotoxin-induced release of tumour necrosis factor (TNF), and of interferon in the circulation of Corynebacterium parvum-treated mice was the subject of this study. TNF was quantified after injection of TNF containing heated serum (TNS) into Meth A sarcoma-bearing mice by determining colour, extent, and incidence of haemorrhagic necrosis. The release of TNF was weakly inhibited by the competitive -blocker phentolamine and the -blocker propranolol. The non-competitive -blocker phenoxybenzamine inhibited to a higher degree. Endotoxin-induced elicitation of growth-inhibiting principles into TNS was antagonized by propranolol and phenoxybenzamine. Administration of adrenaline before endotoxin inhibited the elicitation of TNF and growth-inhibitory activities, which indicates tachyphylaxis. The release of interferon was effectively inhibited by both -adrenoceptor blockers but not by propranolol. The interferon was heat-labile. The results indicate that endotoxin-induced TNF and interferon are separate factors, elicited in different ways. As both -blockers do not only inhibit reactions at the -adrenergic receptor but also reactions at the serotonin receptor and in the case of phenoxybenzamine also at the choline receptor, it is suggested that endotoxin-induced release of the anti-tumour factors is controlled by reactions mediated by one or more of these receptors. It is suggested that the inhibition of TNF release by propranolol may be due to the membrane-stabilizing activity of this agent.     

Salinity dependence,uptake kinetics,and specificity of amino-acid absorption across the body surface of the oligochaete annelidEnchytraeus albidus

Enchytraeus albidus is able to absorb dissolved¹⁴C-labeled neutral amino acids (glycine, L-alanine, L-valine,-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded-aminoisobutyric acid against external glycine or-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non--amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to¹⁴C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,¹⁴C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S).     

A unique example of a pseudo-peptide containing noncoded amino acids self-assembling into a supramolecular β-sheet-like structure in crystals

The crystal structure of a pseudo-peptide with noncoded amino acids (such as N-substituted 3-aminophenylacetic acid and -aminoisobutyric acid) exhibits almost an extended backbone conformation and this pseudo-peptide self-assembled to form an infinite hydrogen bonded, supramolecular, antiparallel -sheet-like structure in solid state.     

Effect of the pH of culture medium on the alkalophilicity of a species of Bacillus

The amino acid incorporation and -aminoisobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8-and 10-bacteria, while no difference was observed in phospholipid composition.Abbreviations AIB Amino-isobutyric acid     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

Inhibition of membrane functions in intact HeLa cells byClostridium difficile cytotoxic culture filtrates

The basis for cytotoxicity to intact HeLa cells by culture filtrates ofClostridium difficile has been investigated. Decrease in intracellular K⁺ levels and inhibition of -aminoisobutyric acid uptake were detected first after exposure to filtrates, followed by inhibition of macromolecular synthesis. Twenty-five percent of the K⁺ remained associated with the cell monolayer, and amino acid uptake and macromolecular synthesis were not totally abolished. These results indicate thatC. difficile culture filtrates preferentially inhibited membrane functions, either by exhausting ATP supplies or by disrupting the permeability barrier of the cell.     

Glutamine and alpha-ketoglutarate uptake and metabolism by nerve terminal enriched material from mouse cerebellum

In order to provide evidence relevant to the hypothesis that nonsynaptically derived -ketoglutarate serves as a metabolic precursor of the neurotransmitter pools of glutamate and GABA the uptake and metabolism of -ketoglutarate by nerve terminal enriched material was studied and compared to corresponding data for glutamine. Both -ketoglutarate and glutamine were transported across the cell membrane by high affinity and low affinity carriers. Under conditions prevailing in vivo -ketoglutarate probably is transported primarily by its high affinity carrier, whereas gluatmine should be transported primarily by one or more low affinity carriers. Based upon reciprocal uptake inhibition experiments glutamine appeared to be transported by the alanine preferring system, and to a lesser extent by the basic amino acid and large neutral amino acid carriers. A comparison of the rate of uptake by different cellular preparations enriched in either nerve terminals or cell bodies indicated that -ketoglutarate is transported selectively by nerve terminals. Both substrates were rapidly converted to glutamate; however, glutamine was more readily metabolized to GABA. The results of our study are consistent with the concept that both glutamine and -ketoglutarate derived from extra-neuronal sources are taken up by nerve terminals and utilized to replenish the neurotransmitter pools of glutamate and GABA.     

Glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1. NeuAc-coupled IL-1 (NeuAc-IL-1) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1 exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1 to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1 was high compared to IL-1. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.     

Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

The evolution of the type I interferon gene family in mammals

A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-, IFN-, and IFN-) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN- diverged first. Although apparent cases of interlocus recombination among mouse IFN- genes were identified, the hypothesis that coding regions of IFN- genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN- were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN- coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN- genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN- and IFN- is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN- genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.     

Synthesis of the 15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Synthesis of zervamicin IIB, specifically labeled at the -position of glutamine-11 with ¹⁵N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered -aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB specifically ¹⁵N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the ¹⁵N-label was clearly detected. The isotope enrichment (98 ± 2%) was determined by FAB-mass spectrometry.     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP31 and MIP32 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP31 and MIP32 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP31 and MIP32 contain four exons and three introns, and MIP31 and MIP32 shared the same intron/exon organization with that of human. The MIP31 and MIP32 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP32 gene was much lower than the Trsc-SCYA107 and MIP31 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP31 and MIP32 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP32 genes in PWBCs was observed at 1–12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP31 expression was observed at 3–12 h poststimulation only with PMA.     

Na+/L+ selectivity in transport systemA: Effects of substrate structure

Summary The effect of amino acid structure on the selectivity between Na and Li as co-substrates for transport SystemA in the Ehrlich cell has been explored to localize relative binding positions. By various tests the relative effectiveness of the two cations varies over fivefold. Changes in structure of the amino acid that lower its response to Na tend to decrease its selectivity for Na over Li, but with many exceptions. The higher the Li level required to half-maximize amino acid entry, the slower tends to be the entry attainable for both Li and amino acid. Our attention fell on strong departures from these trends. An atypically fast uptake is produced by Li in the presence of a second amino group pK₂<8.5, in exceptional association with the known fast uptake in Na. The hydroxyl group of serine yields exceptionally strong uptake, whereas hydroxyl groups in restrained orientation (as in threonine and hydroxyprolines) sharply limit co-substrate interaction. Despite the absence of a sidechain, glycine shows unexceptional relative co-substrate responses. A sidechain in the ₂ position, as ind-alanine, lowers tolerance for both ions, an aberration largely corrected by the insertion of a second (₂) methyl group, and surprisingly, even by an N-methyl group. Forl-alanine, an N-methyl group has in contrast unfavorable effects on co-substrate interaction. These factors point to disturbance by the ₂ methyl group of the position taken by the amino acid at the site, largely rectifiable by balancing effects of a second methyl group. They also point to a position of the alkali ion quite close to the -carbon and far from the position taken in SystemASC. Addition of an ethylene bridge between the -methyl groups of (methylamino)-isobutyric acid leads to the strongest discrimination seen against Li⁺ relative to Na⁺, suggesting through crowding of the area that the alkali ion adjoins the three methyl groups of this analog.     

Primary structure of hemoglobin α-chain from Cuckoo (Eudynamys scolopaceae, Cuculiformes)

The complete amino acid sequence of the ^A-chain of major hemoglobin component from Cuckoo (Eudynamys scolopaceae) is presented. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. The sequence was studied by automatic Edman degradation of the native chain and its tryptic fragments in a gas-phase sequencer. Comparison with other avian hemoglobins shows residues 21, 30, 96, 110, and 114 as being specific to Cuckoo. The functional significance of these is discussed.     

Glutamate and aspartate agonists structurally related to ibotenic acid

Summary This mini-review describes a noval class of excitatory heterocyclic amino acid. The selective interactions of these synthetic amino acids with the central glutamic acid (GLU) and aspartic acid (ASP) receptors have been established on the basis of microelectrophoretic techniques using glutamic acid diethyl ester (GDEE) and -aminoadipic acid (a-AA) as selective antagonists for GLU and ASP, respectively. The parent compound., ibotenic acid (IBO) preferentially activates ASP receptors, but elongation of the side chain of IBO afforded homoibotenic acid (homo-IBO), a GLU agonist. The introduction of bulky substituents into the heterocyclic ring of homo-IBO resulted in a dramatic increase in potency. Alteration of the position of the side chain in IBO to give -amino-5-methyl-3-hydroxy-4-isoxazoleacetic acid (AMAA), preserved the ASP agonism. However, elongation of the side chain of AMAA gave -amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA), which is a very powerful neuronal excitant with selective interaction with the GLU receptors.None of the new compounds are inhibitors of the binding of 3H-kainic acid (³H-KAIN) to rat brain membranes, indicating that the mechanism of action of these compounds is different from that of the neurotoxic compound KAIN. The described compounds may be important tools in future investigations of the physiological role and the mechanism of action of ASP and GLU in the central nervous system (CNS).