Referral patterns of primary care physicians for chronic kidney disease in general population and geriatric patients.

BACKGROUND: Late referral of patients with chronic kidney disease (CKD) by primary care physicians (PCPs) is associated with poor outcomes. We sought to assess factors influencing PCPs referral patterns in the general population and in geriatric patients, and compared their perceptions to the referral patterns. METHODS: We retrospectively reviewed 268 patients with Stage 3 CKD (early referral) and 280 with Stage 4 CKD (appropriate referral) seen in renal clinic and compared them to 268 randomly selected non-referred Stage 4 CKD patients from primary care physicians office records. We also surveyed 400 regional PCPs on factors influencing their referral for CKD. RESULTS: Non-referred patients were significantly (p < 0.05) more likely to be over 65 years (OR: 3.5; 95% CI: 2.3 - 5.2), females (OR: 1.4; 95% CI: 1.0 - 2.0) and of non-white race (OR: 2.6; 95% CI: 1.5 - 4.5) after adjusting for relevant confounding variables. Charlson comorbidity index greater than 4 was associated with non-referral when the non-referral group was compared to the early referral group. Among geriatric patients, women and a higher comorbidity index were associated with non-referral. 25% of PCPs completed the survey and 62% PCPs were unfamiliar with K/DOQI referral guidelines. Age > 75 years, limited life expectancy, patient noncompliance or refusal to consider dialysis influenced PCPs decision to refer. CONCLUSIONS: Our study shows that elderly women, minorities and patients with multiple comorbidities are at risk for non-referral for CKD care. Educating PCPs on the appropriate referral of CKD patients, especially those at risk for late or non-referral to a nephrologist is warranted, as are trials assessing different educational strategies.

Prions affect the appearance of other prions: the story of [PIN(+)].

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN(+)] is required for the de novo appearance of the [PSI(+)] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN(+)]. Thus, [PIN(+)] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN(+)] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.

Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA.

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI+] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI+]-mediated stop codon read-through but do not cure cells of the [PSI+] prion. Interestingly, cells bearing hyg1 seem to enable [PSI+] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI+], is partly relieved.

The relationship between visible intracellular aggregates that appear after overexpression of Sup35 and the yeast prion-like elements [PSI(+)] and [PIN(+)].

Overproduced fusions of Sup35 or its prion domain with green fluorescent protein (GFP) have previously been shown to form frequent dots in [PSI(+)] cells. Rare foci seen in [psi(-)] cells were hypothesized to indicate the de novo induction of [PSI(+)] caused by the overproduced prion domain. Here, we describe novel ring-type aggregates that also appear in [psi(-)] cultures upon Sup35 overproduction and show directly that dot and ring aggregates only appear in cells that have become [PSI(+)]. The formation of either type of aggregate requires [PIN(+)], an element needed for the induction of [PSI(+)]. Although aggregates are visible predominantly in stationary-phase cultures, [PSI(+)] induction starts in exponential phase, suggesting that much smaller aggregates can also propagate [PSI(+)]. Such small aggregates are probably present in [PSI(+)] cells and, upon Sup35-GFP overproduction, facilitate the frequent formation of dot aggregates, but only the occasional appearance of ring aggregates. In contrast, rings are very frequent when [PSI(+)] cultures, including those lacking [PIN(+)], are grown in the presence of GuHCl or excess Hsp104 while overexpressing Sup35-GFP. Thus, intermediates formed during [PSI(+)] curing seem to facilitate ring formation. Surprisingly, GuHCl and excess Hsp104, which are known to promote loss of [PSI(+)], did not prevent the de novo induction of [PSI(+)] by excess Sup35 in [psi(-)][PIN(+)] strains.

Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome.

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6), and 912 (rdn4) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.

Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable.

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.

Dependence and independence of [PSI(+)] and [PIN(+)]: a two-prion system in yeast?

The [PSI(+)] prion can be induced by overproduction of the complete Sup35 protein, but only in strains carrying the non-Mendelian [PIN(+)] determinant. Here we demonstrate that just as [psi (-)] strains can exist as [PIN(+)] and [pin(-)] variants, [PSI(+)] can also exist in the presence or absence of [PIN(+)]. [PSI(+)] and [PIN(+)] tend to be cured together, but can be lost separately. [PSI(+)]-related phenotypes are not affected by [PIN(+)]. Thus, [PIN(+)] is required for the de novo formation of [PSI(+)], not for [PSI(+)] propagation. Although [PSI(+)] induction is shown to require [PIN(+)] even when the only overexpressed region of Sup35p is the prion domain, two altered prion domain fragments circumventing the [PIN(+)] requirement are characterized. Finally, in strains cured of [PIN(+)], prolonged incubation facilitates the reappearance of [PIN(+)]. Newly appearing [PIN(+)] elements are often unstable but become stable in some mitotic progeny. Such reversibility of curing, together with our previous demonstration that the inheritance of [PIN(+)] is non-Mendelian, supports the hypothesis that [PIN(+)] is a prion. Models for [PIN(+)] action, which explain these findings, are discussed.

Characterization of novel rad6/ubc2 ubiquitin-conjugating enzyme mutants in yeast.

Null mutations in the RAD6/UBC2 gene encoding an E2 ubiquitin-conjugating enzyme cause deficiencies in DNA repair, N-end-rule protein degradation, sporulation and telomeric silencing, and alter the preferred integration positions for Ty1 retrotransposons. Here we selected for mutants of RAD6 that cause a release of telomeric silencing. Some alleles retained nearly wild-type ability for sporulation, DNA repair and the degradation of proteins. Alteration in Ty1 integration-site bias accompanied some of these alleles. The possibility that some mutations specifically affect binding of an unknown protein that works with Rad6 in its silencing role, but is not required for DNA repair or N-end-rule activity, is discussed in terms of the Rad6 crystal structure.

Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift.

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.

Host genes that affect the target-site distribution of the yeast retrotransposon Ty1.

We report here a simple genetic system for investigating factors affecting Ty1 target-site preference within an RNAP II transcribed gene. The target in this system is a functional fusion of the regulatable MET3 promoter with the URA3 gene. We found that the simultaneous inactivation of Hir3 (a histone transcription regulator) and Cac3 (a subunit of the chromatin assembly factor I), which was previously shown by us to increase the Ty1 transposition rate, eliminated the normally observed bias for Ty1 elements to insert into the 5' vs. 3' regions of the MET3-URA3 and CAN1 genes. The double cac3 hir3 mutation also caused the production of a short transcript from the MET3-URA3 fusion under both repressed and derepressed conditions. In a hir3Delta single-mutant strain, the Ty1 target-site distribution into MET3-URA3 was altered only when transposition occurred while the MET3-URA3 fusion was actively transcribed. In contrast, transcription of the MET3-URA3 fusion did not alter the Ty1 target-site distribution in wild-type or other mutant strains. Deletion of RAD6 was shown to alter the Ty1 target-site preference in the MET3-URA3 fusion and the LYS2 gene. These data, together with previous studies of Ty1 integration positions at CAN1 and SUP4, indicate that the rad6 effect on Ty1 target-site selection is not gene specific.

The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3.

The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants.

The PNM2 mutation in the prion protein domain of SUP35 has distinct effects on different variants of the [PSI+] prion in yeast.

We have previously described different variants of the yeast prion [PSI+] that can be obtained and maintained in the same genetic background. These [PSI+] variants, which differ in the efficiency of nonsense suppression, mitotic stability and the efficiency of curing by GuHCl, may correspond to different [PSI+] prion conformations of Sup35p or to different types of prion aggregates. Here we investigate the effects of overexpressing a mutant allele of SUP35 and find different effects on weak and strong [PSI+] variants: the suppressor phenotype of weak [PSI+] factors is increased, whereas the suppressor effect of strong [PSI+] factors is reduced. The SUP35 mutation used was originally described as a "Psi no more" mutation (PNM2) because it caused loss of [PSI+]. However, none of the [PSI+] variants in the strains used in our study were cured by PNM2. Indeed, when overexpressed, PNM2 induced the de novo appearance of both weak and strong [PSI+] variants with approximately the same efficiency as the overexpressed wild-type SUP35 allele. Our data suggest that the change in the region of oligopeptide repeats in the Sup35p N-terminus due to the PNM2 mutation modifies, but does not impair, the function of the prion domain of Sup35p.

Yeast Ty1 retrotransposition is stimulated by a synergistic interaction between mutations in chromatin assembly factor I and histone regulatory proteins.

A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition. The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutaions of cac3 and hir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition. We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA. Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3 hir3 double mutant. In addition, the growth rate of the double mutant was reduced. HIR3 belongs to a class of HIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I. Other combinations of mutations in cac and hir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate. A model explaining the synergistic interaction between cac and hir mutations in terms of alterations in chromatin structure is proposed.

Chimeric rRNAs containing the GTPase centers of the developmentally regulated ribosomal rRNAs of Plasmodium falciparum are functionally distinct.

The human malaria parasite, Plasmodium falciparum, maintains at least two distinct types, A and S, of developmentally controlled ribosomal RNAs. To investigate specific functions associated with these rRNAs, we replaced the Saccharomyces cerevisiae GTPase domain of the 25S rRNA with GTPase domains corresponding to the Plasmodium A- and S-type 28S rRNAs. The A-type rRNA differs in a single nonconserved base pair from the yeast GTPase domain. The S-type rRNA GTPase domain has three additional changes in highly conserved residues, making it unique among all known rRNA sequences. The expression of either A- or S-type chimeric rRNA in yeast increased translational accuracy. Yeast containing only A-type chimeric rRNA and no wild-type yeast rRNA grew at the wild-type level. In contrast, S-type chimeric rRNA severely inhibited growth in the presence of wild-type yeast rRNA, and caused lethality in the absence of the wild-type yeast rRNA. We show what before could only be hypothesized, that the changes in the GTPase center of ribosomes present during different developmental stages of Plasmodium species can result in fundamental changes in the biology of the organism.

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion.

[PSI+], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI+]. However, another non-Mendelian determinant, [PIN+], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI+] by Sup35p overproduction in [PIN+] strains, but has no effect on the propagation of [PSI+] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI+] strains. We argue that excess Sup45p inhibits [PSI+] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.

Genetic and environmental factors affecting the de novo appearance of the [PSI+] prion in Saccharomyces cerevisiae.

It has previously been shown that yeast prion [PSI+] is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both [PSI+] and [psi-] yeast strains results in two classes of [psi-] derivatives: Pin+, in which [PSI+] can be reinduced by Sup35p overproduction, and Pin-, in which overexpression of the complete SUP35 gene does not lead to the [PSI+] appearance. However, in both Pin+ and Pin- derivatives [PSI+] is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, [PSI+] curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ [psi-] derivatives are observed in Pin- [psi-] derivatives. Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the [PSI+] prion, is independent of the Sup35p N-terminal domain. A Pin- [psi-] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while [PSI+] curing by Hsp104 overproduction resulted exclusively in Pin+ [psi-] derivatives. We hypothesize that in addition to the [PSI+] prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype.

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.