Distal tubules (DT) from sham or five-sixths nephrectomized (Nx) rats were perfused in vivo to evaluate the hypothesis that, after Nx, endogenous angiotensin II (ANG II) modulates DT in vivo bicarbonate reabsorption (JtCO2) via H(+)-adenosinetriphosphatase (H(+)-ATPase) and Na+/H+ exchange. In Nx rats JtCO2 was increased (65 +/- 7 vs. -24 +/- 21 pmol.min-1.mm-1, P < 0.01). Both luminal and intravenous AT1-receptor blockade by losartan reduced Nx DT JtCO2 (41 +/- 6 and 34 +/- 4 pmol.min-1.min-1, respectively, P < 0.05), whereas neither 10(-9) M nor 10(-11) M ANG II luminal perfusion increased JtCO2, suggesting preexisting high endogenous ANG II levels. The Na+/H+ antiporter inhibitors 5-(N-ethyl-N-isopropyl)-amiloride and 5-(N,N-dimethyl)-amiloride were without effect. Luminal perfusion of 5 nM concanamycin A, a V-type H(+)-ATPase inhibitor, reduced Nx DT JtCO2 (45 +/- 8 pmol.min-1.mm-1, P < 0.05). In Nx A-type intercalated cells, we demonstrated cellular hypertrophy, elaboration of apical microplicae, and enhanced expression/apical polarization of H(+)-ATPase. Thus ANG II is an important determinant in sustaining brisk DT JtCO2 following Nx and is associated with enhanced expression and A-type intercalated cell apical polarization of H(+)-ATPase.
A 1,197-bp region of the broad-host-range plasmid pCUl is adequate for its replication. Analysis of replicating molecules containing this region reveals three clustered origins of vegetative replication and replication proceeds bidirectionally from each in a theta mode. In an Escherichia coli polymerase I deletion mutant, utilization of one of these three origins was not detected. The potentiality for origin utilization may therefore be a determinant of replicon host range.Some groups of plasmids of gram-negative eubacteria are promiscuous relative to other groups (33). Plasmids of the IncN (incompatibility) group are promiscuous and pCUl is a member of this group (11). A PvuII fragment of about 2 kb contains the basic replicon, the sequence features of which have been previously described (20) and are now extended here (see Fig. 1). Previously, a small deletion derivative of pCUl that contained this PvuII fragment was shown to retain the ability to replicate in most hosts (19). Promiscuity is therefore likely to be an attribute of this basic 2-kb replicon. This replicon has another interesting property: although replication and replicon maintenance can occur in vivo in an Escherichia coli poU deletion mutant, two replicon mutants became dependent on functional poLA, implying the presence of alternative pathways of replication and that E. coli polymerase I is essential for only one of the pathways (18). IncN incompatibility was expressed both by the wild-type replicon and the mutants that had becomepolU dependent (17).The purpose of this study was to identify the origin(s) of vegetative replication in this replicon and to ascertain whether origin usage is distinguishable in the presence or absence of functionalpolA. An unexpected result has been the identification of three clustered but distinguishable origins, only two of which are utilized in the poU deletion mutant. These observations are described in this communication, and the results are discussed in the context of plasmid promiscuity.Previously, Krishnan (17) and Narang (26) independently showed that deletions can be made within the 2-kb basic replicon without affecting its ability to replicate. Further deletion analysis of the replicon indicated that all determinants of replication are contained between nucleotides (nt) 195 and 1391 (Fig. 1). The construction of this derivative, pCU996Ql, will be described in detail elsewhere. pCU996fQ contains the segment of DNA (shown as the thickest bar in the pCU996Q1 map in Fig. 1) linked to the fl fragment described by Prentki and Kirsch (28) that confers resistance * Corresponding author.to spectinomycin and streptomycin. pCU996fQ was constructed in E. coli HB101 and then transformed into the nearly isogenic pair of strains SR1758 (also called CM4722) [A(gal bio) thi-J relA1 spoTI] (13, 32) and SR1672 (also called CJ261) [4poU A(gal bio) thi-1 relAl spoTI Kanr] (13, 32). pCU996Q1 was maintained stably in both of these strains when grown for at least 100 generations in Luria broth (without antibiotics) at 37°C at c...
To determine the in vivo effects of chronic ANG II type 1 (AT(1))-receptor blockade by losartan (Los) on enhanced unidirectional bicarbonate reabsorption (J(HCO(3))) of surviving distal tubules, nephrectomized rats drank either water or a solution of Los, 7 days before microperfusion. J(HCO(3)) was suppressed by 50% after Los without further reduction by 5 nM concanamycin A (Conc), suggesting that Los suppresses all Conc-sensitive H(+)-ATPase pumping. Indeed, ultrastructural analysis of A-type intercalated cells revealed a 50% reduction of H(+)-ATPase immunogold labeling of the apical plasma membrane, whereas Western blotting showed that H(+)-ATPase protein levels were also reduced by one-half by Los treatment. To identify other transporters sustaining J(HCO(3)), we perfused three inhibitors simultaneously [5-(N, N-dimethyl) amiloride hydrochloride, Conc, Schering 28080] with or without prior Los treatment: J(HCO(3)) was unchanged despite marked reduction of water reabsorption. We conclude enhanced distal tubule J(HCO(3)) of surviving nephrons is largely mediated by AT(1) receptor-dependent synthesis and insertion of apical H(+)-ATPase pumps in A-type intercalated cells.
Immunological probes were developed to discriminate between a potential biological control fungus and sapstaining fungi present in wood. This paper describes the production of monoclonal antibodies to isolated cell wall fragments of the biological control fungus Gliocladium roseum. Two monoclonals, designated 6A5 and 3F12, were characterized. Their specificity was assessed by ELISA, by immunogold silver staining light microscopy, by immunogold electron microscopy, and by immunoblotting. Monoclonal 6A5 specifically recognized G. roseum and closely related species and did not react with any of 21 sap-staining fungi tested. Monoclonal 3F12 recognized most of the biological control fungi tested and also showed reactivity with two of the 21 sap-staining fungi. Both monoclonals appeared to recognize carbohydrate epitopes of the cell wall in G. roseum. Although the antibodies were produced against the cell wall of fungus grown in liquid culture, they also detected specific fungi in wood and, therefore, can be used for studies of wood colonization by fungi and for investigations of the interactions between different fungi growing on wood.
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