Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum

Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO₃, SO₄ for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO₄OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both¹⁴C-oxalate and³⁵SO₄ were equally sensitive tocis-inhibition by unlabeled SO₄ or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO₄ caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO₃ exchange occur on different carriers because: (i) ClHCO₃ exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO₃ exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO₃ exchanger; (ii) a SO₄OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

The response of the antioxidant defense system in rat hepatocytes challenged with oxysterols is modified by Covi-ox

Suspension cultures of isolated rat hepatocytes were used to investigate whether 7-ketocholesterol and cholestane-3,5,6-triol exert oxidative stress in cells as manifested by increased lipid peroxidation and the induction of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase. The oxysterols were found to increase the levels of both superoxide dismutase and catalase and to have variable effects on glutathione peroxidase activity. Increased lipid peroxidation was not observed, indicating that the endogenous antioxidant defense system was capable of protecting against any oxidative stress that might otherwise by exerted by 7-ketocholesterol or cholestane-3,5,6-triol. Covi-ox, a natural tocopherol blend reduced the effects of both oxysterols on the antioxidant enzymes. A concurrent reduction in the production of thiobarbituric acid-reactive substances in Covi-ox-treated cells is indirect evidence that reactive oxygen species were produced by oxysterols in hepatocyte suspension cultures.     

Ubiquinol regeneration by plasma membrane ubiquinone reductase

Summary Several enzyme systems have been proposed to play a role in the maintenance of ubiquinol in membranes other than the inner mitochondrial membrane. The aim of this study was to investigate the mechanisms involved in NADH-driven regeneration of antioxidant ubiquinol at the plasma membrane. Regeneration was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by high-performance liquid chromatography. Plasma membrane incubation with NADH resulted in the consumption of endogenous ubiquinone, and a parallel increase in ubiquinol levels. The activity showed saturation kinetics with respect to the pyridine nucleotides and was moderately inhibited byp-hydroxymercuribenzoate. Only a slight inhibition was achieved with dicumarol at concentrations reported to fully inhibit DT-diaphorase. Salt-extracted membranes displayed full activity of endogenous ubiquinol regeneration, supporting the participation of an integral membrane protein. In liposomes-reconstituted systems, the purified cytochromeb ₅ reductase catalyzed the reduction of the natural ubiquinone homologue coenzyme Q₁₀ at rates accounting for the activities observed in whole plasma membranes, and decreased the levels of lipid peroxidation. Our data demonstrate the role of the cytochromeb ₅ reductase in the regeneration of endogenous ubiquinol.Abbreviations AAPH 2,2-azobis-(2-amidinopropane) hydrochloride - CoQ coenzyme Q, ubiquinone - CoQH₂ reduced coenzyme Q, ubiquinol - pHMB p-hydroxymercuribenzoate     

Allomonal defence secretions of the American dog tick Dermacentor variabilis (Acari: Ixodidae) promote clustering

The release of the defence secretion from the large wax glands (sensilla sagittiformia) of Dermacentor variabilis ticks modifies the behaviour of other ticks by inducing clustering. A coating of natural tick secretion onto test objects (delipidized ticks, glass beads or filter paper discs) also elicits a clustering response, but a topically applied squalene, its major component, does not have this effect. The clustering response appears to be species specific: D. variabilis cluster on conspecific ticks that secreted but they fail to cluster on Amblyomma americanum or Ixodes scapularis ticks unless coated with secretions from D. variabilis. Volatile components in the defence secretion are involved in recruiting conspecific ticks to those that secreted. When attacked by predatory fire ants, Solenopsis invicta, D. variabilis clustered on individuals that had released the defence secretion. This suggests that the secretion protects ticks from predation by functioning as an alarm pheromone. If confirmed, this is the first report of an alarm pheromone and its glandular source in ticks. The terminology applicable to the integumental glands of ticks is discussed.     

Histochemical and chemical studies on pre- and postnatal development of the different systems of “short” and “long” adrenergic neurons in peripheral organs of the rat

Summary The adrenergic innervation in the submaxillary gland, heart, kidney, small intestine, and accessory male genital organs and the development of the adrenal chromaffin cells and the sympathetic ganglia were studied in the rat from 15 days post coitum to 16 days post partum using the fluorescence histochemical method of Falck and Hillarp. The postnatal development of the noradrenaline concentrations in the heart and vas deferens was followed by fluorometric determinations.At about 15 days post coitum, the anlagen of the sympathetic chains were well visible in the form of two dorsal segmented columns of small branching sympathicoblasts exhibiting an intense catecholamine fluorescence. In the midline, ventrally to these two anlagen, another column of sympathicoblasts developed; this seemed to give rise to the prevertebral ganglia and to the short adrenergic neurons supplying the internal genital organs. At the level of the adrenal anlagen, small intensely fluorescent chromaffin cells were collected in two bilateral groups which became enclosed by adreno-cortical cells. This enclosure was, however, not complete even at two weeks post partum.Bundles of growing sympathetic nerves were visible in the periphery of the various organs studied at 19–21 days post coitum. A terminal innervation of the organs suggestive of a functional transmitter mechanism did not start to establish until at or immediately after birth. The final pattern of innervation was usually reached at about one week post partum, and the following development proceeded largely in the form of a quantitative increase in the number of nerves participating in the innervation apparatus. The adult level of noradrenaline in the heart and vas deferens was reached three to five weeks after birth. The small intestine was an exception in that the final pattern of innervation in the wall was attained immediately after birth.There was no overt difference in the rate of development of the terminal sympathetic innervation in organs supplied by short adrenergic neurons (accessory male genital organs) compared to the innervation of the submaxillary gland, heart and kidney, which receive classical long adrenergic neurons.The work was supported by a grant from the Association for the Aid of Crippled Children, New York, and was carried out within a research organization sponsored by the Swedish Medical Research Council (grants No. B71-14X-56-07A and B71-14X-712-06A).     

Latitudinal gradient of species diversity: multi-scale variability in rocky intertidal sessile assemblages along the Northwestern Pacific coast

This study examined the latitudinal gradient of species diversity of rocky intertidal sessile assemblages on the slopes of rocks along the Northwestern Pacific coast of Japan, located between 31°N and 43°N, by explicitly incorporating an hierarchical spatial scale into the monitoring design. The specific questions were to examine: (1) whether there is a latitudinal gradient of regional diversity, (2) how spatial components of the regional diversity (local diversity and turnover diversity) vary with latitude depending on spatial scale, and (3) whether the latitudinal gradient differs between different measures of species diversity, i.e. species richness and Simpsons diversity index. We measured coverage and the presence or absence of all sessile organisms in a total of 150 census plots established at five shores in each of six regions. The results showed that there were clear latitudinal gradients in regional species richness and in species turnover among shores. However, these patterns were not reflected in smaller-scale local species richness. For Simpsons diversity index, there was no evidence of latitudinal clines either in regional diversity or in spatial components. These results suggest that relative abundance of common species does not vary along latitude, while the number of rare species increases with decreasing latitude.

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An erratum to this article can be found at     

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

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

Interferons up-regulate with different potency HLA class I antigen expression in M14 human melanoma cell line. Possible interaction with glucocorticoid hormones

The relative potency of interferon (IFN), interferon (IFN), and interferon (IFN) in inducing the expression of HLA class I antigens, as well as their capacity to counteract the inhibition induced by glucocorticoid hormones on HLA class I antigen expression, were analysed in the human melanoma cell line M14, both at membrane and at mRNA level. The data obtained indicate that (a) IFN enhance with different potency (IFN>IFN>IFN) the expression of HLA class I antigens in M14 cells, (b) prednisone inhibits HLA class I antigen expresion, (c) glucocorticoid hormones, when associated with IFN or IFN, inhibit the HLA class I enhancement induced by IFN alone, and ffinally, (c) the association between 1 M prednisone or 1 M deflazacort and IFN seems to potentiate the enhancing capacity of IFN on the expression of HLA class I molecules at the mRNA level. These findings, if confirmed, might indicate that IFN and glucocorticoid hormones are not mutually exclusive in the management of human melanoma.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

Morphological stability of Pimpinella anisum hairy root cultures and time-course study of their essential oils

The morphological stability of hairy root cultures of Pimpinella anisum was studied using cultures grown in four different media, both in darkness and under photoperiod conditions of 16 h light; they were first subcultured every eight days and then later on every three weeks. From the four media tested, the hairy root cultures grown in SH medium both in darkness and under photoperiod conditions showed a marked morphological stability with no de-differentiation or greening, when compared with the other culture systems. The time-course study of the essential oils, isolated by distillation-extraction and analysed by GC and GC-MS, showed only quantitative differences in the composition of the oils. A clear heterogeneity in the accumulation pattern was found with regard to the five dominant compounds (pregeijerene, geijerene, zingiberene, -bisabolene and trans-epoxypseudoisoeugenyl 2-methylbutyrate), i.e., those that appeared in a relative amount higher than 5% at least once during the time-course study of the oils from the eight culture systems.     

Polymerization of the Actin Component of the Cytoskeleton and Formation of the Geometry of a Cultured Nerve Cell (a Model Study)

Using mathematical modeling, we studied the biophysical aspects of the growth of the cell membrane and the growth of the actin network of the cytoskeleton of a neuron cultured on the rigid sublayer and the correlation between these processes. To describe the dynamics of the growth of the cytoskeleton limited by the cell membrane, we used the model of the thermal ratchet. Using the approaches of theoretical biophysics, we obtained a simple biophysical criterion that governs the selection of an alternative scenario of the formation of the cell, either growth of a single neurite or growth of a number of neurites. This criterion depends on the value of the adhesion between the cell and the substrate, the dimension of the actin monomer, and the thermal energy determining the frequency of thermal fluctuations of the cell membrane.     

The giant neurone system in ophiuroids

Summary The radial nerve cords of members of the class Ophiuroidea consist of two parts, the ectoneural and the hyponeural tissues, which are separated by an acellular basal lamina. The hyponeural tissue is composed entirely of motor fibres. The cell bodies of the hyponeural neurones are arranged in ganglia, one to each segment of the arm, and each containing approximately one hundred cell bodies. Synaptic contact between the two tissues occurs across the basal lamina. Ultrastructural evidence shows that the majority of these synapses operate in the ectoneural to hyponeural direction. Three pairs of nerve bundles, each containing approximately thirty five large motor fibres arise from each ganglion and innervate the intervertebral muscles. The large motor fibres divide into a number of pre-terminal axons in the region in which the motor fibre enters the muscle block. The terminal axons run at right-angles across the muscle fibres and neuromuscular junctions are found at the points of contact between the two; each terminal axon makes contact with a large number of muscle fibres. The hyponeural axons also pass through the juxtaligamental tissue before they reach the muscle blocks and there is some evidence of synaptic contact with the juxtaligamental cells. The juxtaligamental tissue is thought to be associated with changes in the structural properties of the collagenous ligaments of the arm during arm autotomy (Wilkie 1979). Degeneration studies confirmed the layout of the hyponeural motor axons.     

Mictic patterns of the rotifer Brachionus plicatilis Müller in small ponds

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes (S and L). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study S and L morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.