Studies on the control of sodium excretion in experimental uremia.

A study of the mechanisms governing the high rate of sodium excretion per nephron characteristic of patients with chronic renal disease has been made in dogs. A "remnant kidney" was produced by 85% infarction of the left kidney while the right kidney was left intact. A bladder-splitting procedure allowed simultaneous measurement of glomerular filtration rate and the rate of sodium excretion by each kidney. The animals were fed a constant known amount of sodium chloride and 0.1 mg of 9 alpha-fluorohydrocortisone twice daily throughout the study. In a group of dogs fed 3 or 5 g of salt per day, sodium excretion by the remnant kidney averaged 6.5 muEq/min while the intact kidney was present and 53.7 muEq/min when the animals became uremic after the intact kidney was removed. The increased sodium excretion per nephron by the remnant organ often occurred within 18 hr after contralateral nephrectomy and persisted despite experimentally induced acute reductions in the glomerular filtration rate to below prenephrectomy levels. A second group of animals studied in the same manner but receiving 1 g of salt per day or less failed to develop a natriuresis after contralateral nephrectomy despite high grade uremia. Thus an increased impermeable solute load per nephron was not a regulatory factor in the production of the natriuresis. The increased rate of sodium excretion per nephron in uremia resembles that after saline loading in that it may occur without an increase in glomerular filtration rate or a reduction in mineralocorticoid stimulation. It follows that an additional factor or factors must be involved in the genesis of the natriuresis. In contrast to the natriuresis that is seen in normal animals subjected to saline loading, these uremic animals were found not to have a detectable increase in extracellular fluid volume or blood volume in the presence of high fractional sodium excretion rates. Sodium excretion in response to a small salt load by the remnant organ in uremia was 30% greater than the response of both kidneys in the preuremic state despite a markedly reduced total GFR. These data are consistent with the view that the volume control mechanism becomes more responsive in uremia.

Escherichia coli mut T1. II. Consequences of modification on the association of DNA with the cell membrane.

1. Two isogenic strains of Escherichia coli, K-12 which differ by mutator gene character (mut T1) have been studied. This characteristic caused introduction of a high frequency of undirectional transversions, A-T leads to -CG, into the DNA of the strain harboring it. 2. It had been previously shown that the presence of this gene is accompanied by an alteration of a cell membrane component. Now, the nuclease susceptibility of DNA associated with membrane/DNA/DNA polymerase complexes is reported. DNA of mut T1 membranes is more sensitive towards exogenous nuclease than DNA of membrane complexes from the wild type mut+ strain. 3. Auto-digestion of this DNA by endogenous nuclease associated with the membrane complex is, also, more severe in preparations derived from mut T1 than from the wild-type strain, mut+, but to a lesser extent than observed with exogenous nucleases. 4. Nuclease susceptibility of mut+ membrane bound DNA is markedly influenced by the growth state of the cell. The nuclease susceptibility of membrane bound DNA from mut T1 cells, however, shows no differences between stationary and log states. 5. These differential sensitivities may be due to conformational changes in the membrane introduced as a pleiotrophic consequence of an altered membrane protein. A pertinent role of this protein in a modified replication/repair complex is an attractive suggestion, especially in the context of the mutator character of this strain.

Consequences of modification on the association of DNA polymerase with the cell membrane.

1. This is a report of a possible causal relation between the structure of the membrane-bound DNA polymerase and the mutator characteristic of Exherichia coli, mut T1. The characteristics of the membrane-bound polymerase are compatible with its identification as DNA polymerase II, and enzyme which has been determined to be genetically closely linked to the mut T1 gene. Several genes concerned with membrane structure are also known to be linked to the mutator locus. 2. Differences exist between membrane-bound polymerase activity of a wild-type E. coli K-12 and of an isogenic strain harboring the mutator gene. (a) The cell membranes of the mutator strain retain 5--6 times as much activity as do membrane complexes from wild-type cells. (b) The DNA polymerase activity of the membranes from the mut T1 strain is less sensitive to inhibition by the sulfhydryl-binding reagent, N-ethylmaleimide. (c) The membrane-DNA polymerase complex of mut T1 cells uses endogenous, membrane-bound DNA for replication-repair in preference to exogenous DNA. 3. The differences described are specific to structural differences in the membrane complex. When DNA polymerase activity is solubilized from the complexes, the enzymes of the two strains exhibit similar characteristics. These results are consistent with the thesis that an alteration in membrane structure is the basis of mut T1 activity. The results do not support any hypothesis that mut T1 phenotype is a reflection of mutations in the structural gene for DNA replicase (polymerase) or its components.

Maintenance of DNA integrity during murine erythroleukemia cell differentiation induced by ethionine and other hypomethylation agents.

The extent of single-strand nicks in DNA from murine erythroleukemia cells induced to differentiate to hemoglobin synthesis in the presence of the hypomethylating agent ethionine was estimated and compared to those levels in uninduced cells and from cells induced to differentiate upon exposure to dimethylsulfoxide or butyrate ion. Although ethionine has been shown to cause more extensive hypomethylation in the DNA of induced cells than that caused by dimethylsulfoxide or butyrate ion, the frequency of detected single-strand breaks in the DNA of uninduced, control cells was not significantly different from that of cells exposed to any of these inducing chemicals. This data indicates that no correlation exists between DNA hypomethylation and DNA single-strand breaks and that unmethylated CpG loci likely do not operate as specific endonuclease recognition sites or as potential origins of transcription in these mammalian cells.

Development expression of bone sialoprotein mRNA in rat mineralized connective tissues.

Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein that is a major noncollagenous protein of bone and other mineralizing connective tissues. BSP is characterized by the presence of several polyglutamic acid segments and an RGD motif that mediates cell attachment through a vitronectin-like receptor. Although the precise function of BSP is unknown, the expression of BSP in conjunction with bone formation in vitro indicates a role for this protein in the biomineralization of connective tissues. In this study we used Northern hybridization and in situ hybridization to determine the tissue-specific and developmental expression of BSP during embryogenesis and growth of rat tissues. Analysis of tissues obtained from 13, 17, and 21 day fetuses, and from 4-, 14-, and 100-day-old animals indicates that BSP mRNA expression is restricted to cells actively forming the mineralizing tissues of bone, dentin and cementum. BSP mRNA transcripts were first evident in fully differentiated osteoblasts of 17 day fetal tissues at sites of de novo intramembranous and endochondral bone formation, with maximal expression observed at 21 days of gestation. Thereafter, BSP mRNA levels decreased markedly, and in adult bone hybridization was detected only in the primary spongiosa of long bones. In comparison, mRNAs for osteopontin (OPN), alkaline phosphatase (ALP), and osteocalcin (OC) peaked at 4-14 days postpartum before declining. In the tibiae, Northern hybridization revealed a second peak of mRNA for BSP, ALP, and OPN at 14 days, reflecting an increased osteogenic activity due to the formation of the secondary centers of ossification in the epiphyseal cartilage. In situ hybridization also revealed BSP mRNA in hypertrophic chondrocytes at sites of bone formation, in odontoblasts of the incisor during dentinogenesis, and in cementoblasts during cementogenesis. In view of the restricted distribution and temporal changes in the expression of BSP mRNA that we observed together with the chemical properties of BSP, we believe that this protein has a specific role in mediating the initial stages of connective tissue mineralization.

Characterization of the human bone sialoprotein (BSP) gene and its promoter sequence.

Bone sialoprotein (BSP) is a major structural protein of the bone matrix that is specifically expressed by fully-differentiated osteoblasts. To characterize the gene and to study the tissue-and differentiation stage-specific regulation of BSP gene transcription we have isolated and partially sequenced two overlapping genomic fragments which span the complete human BSP gene and its promoter region. The approximately 15 kb gene comprises seven exons of 82 bp, 68 bp, 51 bp, 78 bp, 63 bp, 159 bp and 2.5 kb (1-7, respectively), separated by six introns of approximately 3 kb, 92 bp, 95 bp, approximately 3 kb, approximately 0.5 kb and approximately 4.5 kb. All of the intron-exon boundaries defining the splice sites conform to the consensus sequence of: AG at the 3' splice site; and GT at the 5' splice site, except the 3' splice site of exon 1. The first exon encodes the 5'-UTR, the second exon the signal sequence and the first two amino acids, exons 3 and 4 the Tyr-and Phe-rich amino terminus, and exon 5 the first segment of polyglutamic acid. Exon 7 encodes over half of the protein including a second polyglutamic acid segment, the RGD cell attachment motif, the sulphated tyrosine-rich C-terminus and the 3'-UTR. The promoter region is characterized by an inverted TATA-like sequence (TTTATA), nts -28 to -23 from the transcriptional start site (+1), and an inverted CCAAT box (ATTGG) at -54 to -50. Analysis of chimeric constructs fused to a CAT reporter gene indicate that the presence of both the inverted TATA-like sequence and CCAAT elements are required for basal promoter activity. Comparison of the human BSP promoter with the rat BSP promoter (Li and Sodek, 1993) reveal that the nature and position of the inverted TATA-like sequence and CCAAT box together with an Ap1 (-148 to -142), CRE (-122 to -116) and a homeobox-binding site (-200 to -191) have been conserved. A putative Glucocorticoid Response Unit (GRU) consisting of a Glucocorticoid Response Element (GRE) and an overlapping direct repeat (DR5) similar to the retinoic acid receptor element (RARE) is present at -1038 to -1022. These studies have defined the structure of the human BSP gene and have identified novel transcriptional elements in the promoter that may be involved in the developmentally regulated, tissue specific expression of this gene.

Evolution of a series of peptidoleukotriene antagonists: synthesis and structure/activity relationships of 1,3,5-substituted indoles and indazoles.

1,3,5-Substituted indoles and indazoles have been studied as receptor antagonists of the peptidoleukotrienes. The best of these compounds generally had a methyl group at the N1 position, a [(cyclopentyloxy)carbonyl]amino or 2-cyclopentylacetamido or N'-cyclopentylureido group at the C-5 position, and an arylsulfonyl amide group as part of the acidic chain at the C-3 position of the ring. Such compounds had in vitro dissociation constants (KB) in the range 10(-9) - 10(-11) M on guinea pig trachea against LTE4 as agonist and inhibition constants (Ki) less than or equal to 10(-9) M on guinea pig parenchymal membranes against [3H]LTD4. A number of compounds were orally effective at doses less than or equal to 1 mg/kg in blocking LTD4-induced "dyspnea" in guinea pigs. Compound 45 [N-[4-[[5-[[(cyclopentyloxy)carbonyl]-amino]-1-methylindol-3- yl]methyl]-3-methoxybenzoyl]-2-methylbenzenesulfonamide, ICI 204,219; pKB = 9.67 +/- 0.13, Ki = 0.3 +/- 0.03 nM, po ED50 = 0.3 mg/kg] is currently under clinical investigation for asthma. In the indole series, certain alkylsulfonyl amides possessing a 3-cyanobenzyl substituent at the N-1 position (60, 61) were produced that had KB less than or equal to 10(-9) M on guinea pig trachea.

Synthesis and in vitro LTD4 antagonist activity of bicyclic and monocyclic cyclopentylurethane and cyclopentylacetamide N-arylsulfonyl amides.

The dissociation constants (KB) at the LTD4 receptor on guinea pig trachea of a series of monocyclic and bicyclic cyclopentylurethane and cyclopentylacetamide N-arylsulfonyl amides have been measured. The KB was found to be remarkably tolerant of changes in the electronic constitution and lipophilicity of the bicyclic ring system (template). Thus, N-[4[[6-[[(cyclopentyloxy)carbonyl]amino]benzimidazol-1- yl]methyl]-3-methoxybenzoyl]benzenesulfonamide (11a) and N-[4-[[5-[[(cyclopentyloxy)carbonyl]amino]benzo[b]thien-3- yl]methyl]-3-methoxybenzoyl]benzene-sulfonamide (25a) had closely similar affinities (pKB, 9.20 and 9.31, respectively; LTE4 as agonist). It has been shown that the hetero-ring of the template need not be aromatic in order to achieve high affinity, since indoline 31 and 2,3-dihydrobenz-1,4-oxazines 37a-c had pKBs greater than 9. Further, it has been shown that an o-aminophenone (see 42 and Figure 3) can function as a template; the template in 42 [see iii] is bicyclic by virtue of the presence of an intramolecular hydrogen bond. In contrast, when the template is a phenyl ring (48), receptor affinity is markedly reduced. These findings support the notion that central bicyclic ring system in this family of peptidoleukotriene antagonists is a molecular feature which helps to preorganize the acylamino and acidic chains and thereby facilitate the molecular recognition event.

Hydroxyacetophenone-derived antagonists of the peptidoleukotrienes.

Considerations of the possible similarities between leukotriene D4 and its prototypical antagonist, FPL 55712, led to the development of a new series of leukotriene antagonists incorporating a hydroxyacetophenone group (e.g., the toluic acids 16 and 18). Although considerable attention has focused on FPL 55712-derived analogues, only limited investigations into alternatives for the standard 4-acetyl-3-hydroxy-2-propylphenoxy moiety have been reported. Therefore, an extensive study of modifications to the hydroxyacetophenone portion of toluic acid 18 was undertaken. Although no viable alternative to the 3-hydroxy moiety was discovered, replacements for the 2-propyl group (34, 37) and the 4-acetyl functionality (56, 59) yielded potent antagonists. A number of compounds exhibited longer duration of action in vivo than FPL 55712.

N-aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamides: KATP potassium channel openers. Modifications on the western region.

A subset of antiandrogen compounds, the N-aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamides 1, were found to activate ATP sensitive potassium channels (KATP) and represent a new class of potassium channel openers (PCOs). A structure-activity relationship was carried out on the western region of this series with the goal of obtaining an activator of the ATP sensitive potassium channel suitable for use in the treatment of urge urinary incontinence. In particular three large 4-(N-aryl) substituents, the (N-phenyl-N-methylamino)sulfonyl, benzoyl, and 4-pyridylsulfonyl moieties, yielded non-antiandrogen, KATP potassium channel openers (39, 41, and 64, respectively) that are bladder selective in an in vivo rat model that simultaneously measures bladder contractions, heart rate, and blood pressure. Substitutions of the aryl rings of 41 and 64 gave several derivatives that also display selectivity in the in vivo rat model; however, none appear to offer a substantial advantage over 41 and 64. The PCO activity of 41 and 64 resides in the (S)-(-) enantiomers. ZD6169, 41(S), has been selected into development for the treatment of urge urinary incontinence.

Minimal renal amyloidosis with nephrotic syndrome.

Two patients older than age 50 years had minimal amyloidosis in association with the nephrotic syndrome. The amyloid deposits in each case were so inconspicuous as to have been initially overlooked, and both biopsy specimens were thought to show minimal glomerular changes. A few, widely scattered, silver-positive, epimembranous spicules were found on reexamination by light microscopy, and Congo red and thioflavin T stains and electron microscopy confirmed the presence of small glomerular amyloid deposits. Both patients have since died of renal failure. In our series of biopsy specimens, amyloidosis was found in 14.2% of patients older than age 50 years presenting with nephrotic syndrome or severe proteinuria, over 1 1/2 times the frequency of minimal-change nephrotic syndrome. We therefore urge careful examination for amyloid deposits of all kidney biopsy specimens with the appearance of minimal-change nephrotic syndrome in older patients. Fluorescence microscopy of Congo red- and thioflavin T-stained sections is very helpful in the detection of small deposits.

Studies on the nucleotide arrangement in DNA. Estimation of sequence isomers in pyrimidine deoxyribo-oligonucleotides.

The population of pyrimidine deoxyribo-oligonucleotides obtained from DNA may be separated, first, into fractions comprising identical lengths (isotichs) and, subsequently, into the individual components of an isostich fraction varying in the cytidylic and thymidylic acid composition. At a third level of analysis the proportion of components exhibiting the same composition but different sequence may be determined. Selective removal of cytosine and the subsequent analysis for characteristic thymidine derivatives released during an acid-catalyzed beta-elimination reaction allowed estimation of the relative proportion of sequence isomers for pyrimidine isostichs of length two to five.

Chemical inducers of differentiation cause conformational changes in the chromatin and deoxyribonucleic acid of murine erythroleukemia cells.

The chemical inducers of murine erythroleukemia cell differentiation, dimethyl sulfoxide, sodium butyrate, and ethionine, elicited conformational changes in the DNA and chromatin of treated cells. The chromatin from dimethyl sulfoxide treated and butyrate-treated cells exhibited circular dichroic spectra different from that of the noninduced control. The molar ellipticity [theta]282.5 in isotonic saline decreased from 4900 deg X cm2 X dmol-1 for control chromatin to 3800 and 3600 deg X cm2 X dmol-1 for dimethyl sulfoxide treated and butyrate-treated chromatin, respectively, while that from ethionine-treated chromatin remained virtually unchanged (5400 deg X cm2 X dmol-1). Increasing the ionic strength to 2.5 or 5 M with NaCl resulted in a substantial, uniform, decrease in molar ellipticity. Thermal denaturation profiles of high molecular weight DNA prepared from each of the inducer-treated cells showed a pronounced hyperchromic shift but no change in Tm when compared to control DNA. Circular dichroic spectra of the DNA indicated a decrease in ellipticity [theta]277 from 9600 deg X cm2 X dmol-1 to 8900, 8300, and 8800 deg X cm2 X dmol-1 for ethionine, dimethyl sulfoxide, and butyrate treated cells, respectively. Treatment of the DNA with 3 M NaCl canceled the UV and CD differences. These measurements indicate an increased stacking of bases or an increased compactness of the DNA from induced cells. Concomitant with specific modifications such as hypomethylation of DNA, the data can be interpreted in terms of conformational changes in chromatin resulting from core histone acetylation.

Controlled comparison of gentamicin and tobramycin nephrotoxicity.

317 patients with suspected or documented infections other than cystitis were randomly assigned to receive gentamicin or tobramycin dosed according to the Sawchuk/Zaske method or a modification of the McHenry method. 196 patients completed 6 days of therapy, had serial determinations of serum creatinine and were evaluated for nephrotoxicity. Within each dosing method group those patients receiving gentamicin and tobramycin had a similar mean age, initial serum creatinine level, total dose, duration of therapy and trough serum aminoglycoside concentration. Nephrotoxicity developed in 5 of 49 (10.2%) given gentamicin via the McHenry method compared to 9 of 49 (18.4%) given tobramycin. Similarily, 4 of 50 (8.0%) given gentamicin via the Sawchuk/Zaske method developed nephrotoxicity compared to 8 of 48 (16.7%) given tobramycin. Within the Sawchuk/Zaske and the modified McHenry dosing method groups, no significant difference was noted in the incidence of nephrotoxicity associated with gentamicin and tobramycin. 34% of patients with elevated trough serum aminoglycoside concentrations developed nephrotoxicity compared to 3.7% of those with nonelevated troughs (p less than 0.0005).

Characterization of porcine bone sialoprotein: primary structure and cellular expression.

Bone sialoprotein (BSP) is a highly glycosylated and sulphated phosphoprotein that is a major non-collagenous protein of bone. To further characterize the porcine protein and to study its expression during bone formation BSP cDNA clones were isolated from a porcine bone cDNA library. The primary sequence of the protein was derived from the nucleotide sequence of the largest cDNA insert and from the amino-terminal amino acid sequence determined by the automated Edman degradation procedure. When compared with sequences obtained from the human and rat BSPs 74% and 64% of the amino acids, respectively, were identical and a further 11% and 17%, respectively, were conservative replacements. Moreover, 60% of the amino acids in a concensus sequence derived from the primary sequences of mammalian BSPs were conserved with 16% conservative replacements. The two stretches of polyglutamic acid, through which the protein is capable of binding to hydroxyapatite, and an RGD motif that mediates cell attachment are retained in conserved sequences as are a number of potential sites of serine, threonine and tyrosine phosphorylation, glycosylation and tyrosine sulphation. Secondary structure prediction and hydrophilicity analysis indicate that the nascent BSP has an open flexible structure with the potential to form significant amounts of alpha-helix and some beta-sheet. In situ hybridization of fetal porcine bone with cRNA probes to porcine BSP mRNA shows that BSP is specifically expressed in differentiated osteoblasts on the surface of newly-forming bone trabeculae with especially high levels of hybridization at sites of de novo bone formation. The highly conserved features of BSP and its restricted distribution indicate an important role for this sialoprotein in the formation of bone.

Localization of bone sialoprotein (BSP) expression to sites of mineralized tissue formation in fetal rat tissues by in situ hybridization.

Bone sialoprotein (BSP) is a major protein in the mineralized matrix of bone and dentine. To study the relationship between the expression of BSP and the formation of mineralized connective tissues, a cDNA probe to rat BSP was prepared for in situ hybridization analysis of developing fetal rat bones and teeth. When used for Northern hybridization analysis of rat bone marrow cells induced to differentiate into osteogenic cells by dexamethasone, the BSP cDNA revealed a specific induction of 1.6- and 2.0-kb mRNA species of BSP. In tissue sections a strong hybridization signal associated with osteoblasts was observed in areas of endochondral bone formation in the long bone metaphysis and condylar cartilage, and in the intramembranous bone of the calvaria and mandible. Hybridization reflecting a lower degree of expression was evident in cells of the transitional zone of mineralizing cartilage and in odontoblasts forming incisor dentine. Expression of BSP was also demonstrated in the hypertrophic cartilage cells in the long bone and condylar process. In contrast, expression of BSP could not be detected in the reserve or proliferative chondrocytes, fibroblasts and muscle cells. These studies demonstrate that the expression of BSP in bones and teeth is essentially restricted to cells directly involved in the formation of mineralizing connective tissue matrices, indicating that BSP has a specific role in biological mineralization and that it is a useful marker of bone formation.