Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Synthesis of polypeptides by microwave heating II. Function of polypeptides synthesized during repeated hydration-dehydration cycles

Summary Polypeptides, synthesized from a mixture of amino acid amides by microwave heating during repeated hydration-dehydration cycles, showed hydrolase- and oxidoreductase-like catalytic activities. Poly(GAVDH), polypeptides synthesized from an equimolar mixture (each 0.1 M) of glycinamide,l-alaninamide,l-valinamide,l-aspartic acid -amide, andl-histidinamide, catalyzed the hydrolysis of PNPAc. The hydrolytic rate of PNPAc with poly(GAVDH) was the quadruple of that ofl-histidine alone. Though the k_cat values of different resulting polypeptides were 10³–10⁶ times less than those of native hydrolases, the K_m value of the polypeptides further containing phenylalanine residues was nearly equal to that of the esterase. This result indicates the presence of hydrophobic interaction between a substrate and the polypeptides. Resulting polypeptides also showed catalytic activity for the reduction of ferricyanide ion [Fe(CN)^3–] with NADH. The polypeptides seemed to have a strong affinity for adenine nucleotides, because the reaction was inhibited by adenine derivatives such as NAD⁺ and AppA. A hypothesis for the emergence of primitive protein enzymes is discussed.     

Interactions Between Calonectria crotalariae and Heterodera glycines on Soybean

The interactions of Heterodera glycines at four egg inoculum levels (0, 100, 1,000, and 10,000 per pot) and three cyst levels (0, 100, and 200 per pot) and Calonectria crotalariae at 500, 5,000, and 50,000 microsclerotia per pot were evaluated on soybean. At the two lowest nematode egg levels, the presence of C. crotalariae did not affect nematode reproduction. At 10,000 eggs per pot, however, nematode reproduction was increased significantly at each microsclerotial level. The increase in nematode reproduction was stepwise at 500 and 5,000 microsclerotia per pot but declined at 50,000 microsclerotia per pot. Similar results were obtained when cysts rather than eggs were used as nematode inoculum. The nematode x fungus interaction significantly affected 60-day plant growth parameters of both Lee 74 and Centennial soybean. The nematode x fungus interaction was antagonistic to plant roots and significantly influenced root injury ratings. The presence of C. crotalariae in tissues of stock plants or plants used as race differentials did not alter the analysis of this population as race 3.     

Synthesis of polypeptides by microwave heating I. Formation of polypeptides during repeated hydration-dehydration cycles and their characterization

Summary Amino acid amides effectively reacted to produce polypeptides in response to microwave heating during repeated hydration-dehydration cycles. The polypeptides, formed from a mixture of glycinamide, alaninamide, valinamide, and aspartic acid -amide, had molecular weights ranging from 1000 to 4000 daltons. Amino acids were incorporated into the polypeptides in proportion to the starting concentrations, with the exception of glycine whose incorporation was 1.5 times higher than that of the other amino acids. The polypeptides had some definite secondary structure, such as -helix and -sheet, in aqueous solution. This reaction provides not only a convenient method for abiotic peptide formation but also a convenient method for the chemical synthesis of peptides.     

Molecular Diagnosis of Meloidogyne Species

Genetic variation within nuclear and mitochondrial DNA of Meloidogyne species and host races has been evaluated for the development of root-knot nematode molecular diagnostics. This review summarizes the distinctive features of several useful DNA-based assays for plant-parasitic nematodes, focusing upon the direct application of these procedures for Meloidogyne detection, identification, and systematics.     

Phylogenetic Implications of Phasmid Absence in Males of Three Genera in Heteroderinae

Absence of the phasmid was demonstrated with the transmission electron microscope in immature third-stage (M3) and fourth-stage (M4) males and mature fifth-stage males (M5) of Heterodera schachtii, M3 and M4 of Verutus volvingentis, and M5 of Cactodera eremica. This absence was supported by the lack of phasmid staining with Coomassie blue and cobalt sulfide. All phasmid structures, except the canal and ampulla, were absent in the postpenetration second-stage juvenile (J2) of H. schachtii. The prepenetration V. volvingentis J2 differs from H. schachtii by having only a canal remnant and no ampulla. This and parsimonious evidence suggest that these two types of phasmids probably evolved in parallel, although ampulla and receptor cavity shape are similar. Absence of the male phasmid throughout development might be associated with an amphimictic mode of reproduction. Phasmid function is discussed, and female pheromone reception ruled out. Variations in ampulla shape are evaluated as phylogenetic character states within the Heteroderinae and putative phylogenetic outgroup Hoplolaimidae.     

Development of Bursaphelenchus xylophilus Populations in Wood Chips with Different Moisture Contents

Bags of Pinus strobus wood chips with moisture contents of 38, 92, 164, and 217% (oven dry weight) were inoculated with Bursaphelenchus xylophilus and incubated at 30 C in order to determine the effect of wood moisture on nematode population development. Nematodes were extracted after 2, 4, 8, and 12 weeks. Population levels were greatest in wood chips with a moisture content of 38% and decreased successively with each higher moisture content. In chips with the three lower moisture contents, populations peaked at 2 weeks, but at 217% moisture, they peaked at 8 weeks. By 12 weeks, nematode populations had declined in wood chips with 92 and 164% moisture contents. The fungi most frequently isolated from the wood chips were Alternaria, Fusarium, Gliocladium, Graphium, Penicillium, Trichoderma, and Mucorales.     

2''-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery

2'-Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 microM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 micrograms/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the greater than 95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5-50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68-78% of control values in brain regions with normally high levels of activity and by 44-59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.