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D S Goldfarb

Publications and source records attributed to D S Goldfarb.

At least 55 records · Page 3Linked to original sources
PubMed1993

Most nuclear proteins are imported by a single pathway.

Using kinetic competition analysis, we show that the import of most Xenopus oocyte nuclear proteins is directed by a single family of nuclear localization signals (NLS). The transport of microinjected [35S]methionine-labeled nuclear proteins into oocyte nuclei was challenged with saturating concentrations of SV40 large T-antigen NLS peptide-BSA (P(Lys)-BSA) and was assessed by SDS-acrylamide gel electrophoresis and fluorography. P(Lys)-BSA was effective at competing the import of virtually all larger polypeptides but had a less marked effect on the mediated import of many smaller polypeptides. Wheat germ agglutinin, an inhibitor of nuclear pore complex function, enhanced the competitive effects of P(Lys)-BSA on the import of both large and small polypeptides. These data indicate that most 35S-labeled Xenopus oocyte nuclear proteins, including nucleoplasmin, contain NLSs that are functionally related to the T-antigen NLS and probably employ the same cytosolic NLS receptor(s).

Amino Acid Sequence↗
PubMed1992

NIP1, a gene required for nuclear transport in yeast.

Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.

Alleles↗
PubMed1991

Effects of pH and cyclic adenosine monophosphate on ileal electrolyte transport in the rat and rabbit.

Alterations in extracellular pH cause reciprocal changes in NaCl absorption in the rat and rabbit ileum. The presence of cholera toxin-induced secretion does not affect pH action measured by in vivo perfusion of the rat ileum. We examined the interaction of pH and cyclic adenosine monophosphate-induced secretion in the rabbit ileum. We found that alterations in arterial pH did not affect ileal absorption in the rabbit in the presence of cholera toxin-induced secretion. This was true whether transport was studied during in vivo ileal perfusion of anesthetized rabbits or by measuring Na+ and Cl- fluxes across isolated, short-circuited tissues in the Ussing chamber. The effects of pH also were blocked when normal rabbit ileum was exposed to 1 mmol/L dibutyryl cyclic adenosine monophosphate in vitro. By contrast, alterations in bathing solution pH affected ileal absorption in the rat in the presence and absence of cyclic adenosine monophosphate. Similarly, exposure to cyclic adenosine monophosphate did not affect the response of the rat colon to PCO2. These findings suggest that the apparently independent effects of pH and cyclic adenosine monophosphate in the rat ileum are not universal. In tissues such as the rabbit ileum, the mechanisms of pH and cyclic adenosine monophosphate action may have biochemical or physiological pathways in common.

Animals↗
PubMed1991

Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway.

Protein import to the nucleus is a signal-mediated process that exhibits saturation kinetics. We investigated whether signal bearing proteins compete with U2 and U6 snRNPs during import. When injected into Xenopus oocytes, saturating concentrations of P(Lys)-BSA, a protein bearing multiple nuclear localization signals from SV40 large T-antigen, reduce the rate of [125I]P(Lys)-BSA and of [125I]nucleoplasmin import, consistent with their competing for and sharing the same limiting component of the import apparatus. In contrast, saturating concentrations of P(Lys)-BSA do not reduce the rate of HeLa [32P]U2 snRNP assembly or import. The import of U6 snRNP is also competed by P(Lys)-BSA. We conclude that U2 snRNP is imported into oocyte nuclei by a kinetic pathway that is distinct from the one followed by P(Lys)-BSA, nucleoplasmin, and U6 snRNP.

Animals↗
PubMed1991

Effect of thiazides on colonic NaCl absorption: role of carbonic anhydrase.

The mechanisms by which the benzothiadiazide class of diuretics inhibit electroneutral NaCl absorption are not fully understood. We studied the mechanisms of thiazide action in perfused loops of distal colon in anesthetized male Sprague-Dawley rats. Hydroflumethiazide (1 mM) reversibly inhibited greater than 40% of Na, Cl, and water absorption. Prior exposure of the colon to the carbonic anhydrase inhibitor methazolamide (0.1 mM) prevented the effects of hydroflumethiazide and metolazone, a thiazide-like drug, on colonic absorption. In Ussing flux chambers, addition of hydroflumethiazide to both the mucosal and serosal bathing solutions (but not to the mucosal solution alone) caused marked decreases in Na and Cl absorption. Such inhibition only occurred at concentrations of hydroflumethiazide (0.1 and 1.0 mM) that inhibited greater than 90% of carbonic anhydrase activity in homogenized colonic mucosa. We conclude that an important mechanism by which thiazides inhibit NaCl absorption in the rat distal colon is by inhibition of mucosal carbonic anhydrase. In tissues containing this enzyme, this mechanism of thiazide effect on ion flux must be considered.

Acetazolamide↗
PubMed1990

Facilitated nuclear transport of histone H1 and other small nucleophilic proteins.

Upon microinjection into the cytoplasm, three small nonnuclear (extracellular or mitochondrial) proteins diffused into nuclei of chilled or energy-depleted cells. In contrast, the facilitated transport of two large nuclear localization signal (NLS)-containing proteins was reversibly arrested by chilling or energy depletion. Surprisingly, the transport of two small nucleophilic proteins, histone H1 and P(Lys)-cytochrome c (cytochrome c cross-linked with synthetic peptide NLSs), was also arrested by either chilling or energy depletion. In situ titration studies indicate that the transport arrest of H1 in chilled cells is mediated by a cytoplasmic receptor. Therefore, even though they are potentially able to diffuse into nuclei, histones and other small NLS-containing proteins are localized by a receptor-mediated process that precludes their diffusion through the nuclear pores.

Amino Acid Sequence↗
PubMed1989

Nuclear transport.

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Amino Acid Sequence↗
PubMed1989

Protein import through the nuclear pore complex is a multistep process.

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.

Acetylglucosamine↗
PubMed1989

Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei.

Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila. Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating. The way in which histone H4 localized to either the macronucleus or the micronucleus suggested that it accumulates in whichever nucleus is replicating. The inability of the micronucleus to accumulate Tetrahymena H1 or heterologous nuclear proteins, even at a period in the cell cycle when it is accumulating H4, suggests that it has a specialized transport system. These studies demonstrate that although the mechanism for localizing proteins to nuclei is highly conserved among eukaryotes, it can differ between two porecontaining nuclei lying in the same cytoplasm.

Animals↗
PubMed1988

Effects of acid-base variables on ion transport in rat colon.

Alterations in arterial acid-base variables have important effects on colonic electrolyte transport in vivo. To confirm the relative effects of these variables and to characterize the transport processes involved, we measured unidirectional 22Na and 36Cl fluxes across short-circuited, distal colonic mucosa of Sprague-Dawley rats. Stripped tissues were studied in Hepes buffer and in Ringer's solutions at HCO3 concentrations of 11, 21, and 39 mM, and CO2 tensions between 0 and 69.6 mmHg. Increases in PCO2, but not in either pH or HCO3 concentration, caused similar increases in JNanet and JClnet (net flux of sodium and chloride, respectively) from -0.2 +/- 0.3 and -1.5 +/- 0.4 mu eq/cm2 per h at PCO2 = 0 to 6.8 +/- 0.6 and 7.6 +/- 0.7 mu eq/cm2 per h, respectively, at PCO2 = 69.6 mmHg. These increases were accounted for by changes in Jms and were accompanied by small decreases in Isc. 1 mM acetazolamide decreased both JNanet and JClnet and their responses to increases in CO2. 0.75 mM luminal amiloride prevented the increase in sodium absorption, but did not affect the CO2-induced increase in chloride absorption. In the presence of amiloride, CO2 increased JR (residual flux). 0.1 mM luminal furosemide did not affect the CO2-induced increases in JNanet in the absence or presence of amiloride. Changes in HCO3 concentration did not alter JR. We conclude that ambient CO2 effects active, electroneutral sodium absorption in the rat distal colon. The process stimulated by CO2 is dependent on mucosal carbonic anhydrase activity and most likely represents Na/H and Cl/HCO3 ion exchange.

Acetazolamide↗
PubMed1987

Effect of systemic acid-base balance on ileal secretion.

We previously reported that systemic pH and HCO3 concentration affect ileal water and electrolyte absorption. To determine whether these effects could influence an ongoing secretory process, we measured transport in ileal loops exposed to either saline or 50-75 micrograms cholera toxin in mechanically ventilated Sprague-Dawley rats anesthetized with pentobarbital sodium. The effects of acute respiratory and metabolic acidosis and alkalosis were then examined. Decreases in systemic pH during respiratory acidosis caused equivalent increases in net water (54 +/- 8 microliters . cm-1 . h-1) and Na absorption (7 +/- 1 mu eq . cm- . h-1) and smaller increases in Cl absorption in cholera toxin compared with saline loops. These increases reversed the net secretion of these ions observed during alkalemia in the cholera toxin loops to net absorption. Metabolic acidosis and alkalosis and respiratory compensation of systemic pH of these metabolic disorders also altered cholera toxin-induced secretion in a direction consistent with the pH change. The increase in net HCO3 secretion caused by cholera toxin was unaffected by the respiratory disorders and did not vary with the HCO3 concentration in the metabolic disorders. These findings suggest that the systemic acid-base disorders that characterize intestinal secretory states may themselves alter intestinal absorptive function and fluid losses.

Acid-Base Equilibrium↗
PubMed1985

Mutations that affect the translation efficiency of Tn9-derived cat gene in Bacillus subtilis.

We have isolated two spontaneous mutations that increase the expression of the Tn9-derived cat gene in Bacillus subtilis. These mutations, which appear to affect initiation of translation of chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) consist of a tandem duplication and triplication of a 55-base-pair sequence located at the 5' end of cat. Included in the repeated sequence are the Shine-Dalgarno site, initiation codon, and a region of dyad symmetry located within the structural portion of the cat gene. A striking feature of the mutated initiation sites is their potential to form stem-loop structures at the 5' end of the cat messenger RNA. Within the single-stranded loops of these structures are the ribosome binding site and initiation codon for the cat gene. It appears that the Gram-negative cat translation initiation site has mutated to permit efficient utilization in B. subtilis without directly affecting Shine-Dalgarno sequence homology. This report suggests that secondary structure in the vicinity of the Shine-Dalgarno site can exert a strong positive influence on the initiation of translation in B. subtilis.

Acetyltransferases↗
PubMed1984

The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo.

A cloned Bacillus subtilis gene (sprE) expressed only during the stationary growth phase is shown to encode the subtilisin E protease, an enzyme associated with sporulation. We have determined the DNA sequence of the sprE promoter region and the promoter-proximal half of the structural gene. The sprE gene codes for a putative 29-residue signal peptide and a 77-residue leader peptide preceding the mature subtilisin sequence. By plasmid integration and phage PBS1 transduction, we have mapped the sprE locus between glyB and metD on the B. subtilis chromosome, a region also containing the hyperprotease-producing hpr gene. In vitro the sprE gene is transcribed by the minor form of RNA polymerase containing a 37,000-dalton sigma factor (sigma 37). We show by S1 nuclease mapping that sprE transcription initiates at dual start sites both in vitro and in vivo and that the promoter for the downstream site has a characteristic sigma 37 recognition sequence. We propose that the physiological role of the sigma 37 RNA polymerase is to transcribe a class of genes that are catabolite repressed, that encode extracellular enzymes, or that are expressed only during the stationary phase of growth.

Amino Acid Sequence↗
PubMed1983

A temporally regulated promoter from Bacillus subtilis is transcribed only by an RNA polymerase with a 37,000 dalton sigma factor.

A 1,250 base pair Bacillus subtilis chromosomal HindIII restriction fragment (S fragment) has been cloned into the B. subtilis expression-probe plasmid pGR71. The S fragment induces the expression of the pGR71 chloramphenicol resistance gene shortly after the initiation of sporulation. The transcriptional promoter responsible for the expression of this temporally regulated genetic element has been identified and mapped in vitro. This promoter is recognized exclusively by the minor B. subtilis RNA polymerase that contains the 37,000 dalton sigma factor.

Acetyltransferases↗