Biomedical subjects

A D Johnson

Publications and source records attributed to A D Johnson.

At least 19 recordsLinked to original sources

PubMed2005

Mating in Candida albicans and the search for a sexual cycle.

Candida albicans is a normal part of the human microflora, but it is also an opportunistic fungal pathogen that causes both mucosal infections and life-threatening systemic infections. Until recently, C. albicans was thought to be asexual, existing only as an obligate diploid. However, a mating locus was identified that was homologous to those in sexually reproducing fungi, and mating of C. albicans strains was subsequently demonstrated in the laboratory. In this review, we compare and contrast the mating process in C. albicans with that of other fungi, particularly Saccharomyces cerevisiae, whose mating has been most intensively studied. Several features of the mating pathway appear unique to C. albicans, including aspects of gene regulation and cell biology, as well as the involvement of "white-opaque" switching, an alteration between two quasi-stable inheritable states. These specializations of the mating process may have evolved to promote the survival of C. albicans in the hostile environment of a mammalian host.

Animals↗

PubMed2002

Opioid circuits originating from the nucleus paragigantocellularis and their potential role in opiate withdrawal.

Neurons in the rat nucleus paragigantocellularis (PGi), located in the ventrolateral medulla, send collateral projections to the locus coeruleus (LC) and to the nucleus of the solitary tract (NTS). The present study examined whether neurons in the PGi that project to both the LC and NTS contain leucine(5)-enkephalin (ENK), and also whether opioid-containing neurons in the PGi are activated following withdrawal from opiates. Retrograde transport of Fluoro-Gold (FG) from the LC and transport of a protein-gold tracer from the NTS was combined with detection of an antibody directed against ENK in the PGi. Using fluorescence and brightfield microscopy, it was established that more than half of the neurons containing both FG and the protein-gold tracer, also exhibited immunolabeling for ENK. The most frequent location of triply labeled neurons was the retrofacial portion of the PGi. In a separate series, rats were chronically implanted with morphine or placebo pellets and, on the fifth day, were subjected to an intraperitoneal injection of naltrexone. Two hours following initiation of withdrawal, rat brains were obtained and processed for detection of c-fos and in situ hybridization labeling of preproenkephalin (PPE) mRNA. Naltrexone injections into morphine-dependent rats caused a dramatic increase in c-fos as compared to control rats. Approximately 66% of the c-fos-labeled neurons exhibited labeling for PPE mRNA. These were also enriched in the retrofacial portion of the PGi. Taken together, the present data indicate that withdrawal from opiates engages opioid neurons in the PGi, some of which may coordinate activity of neurons in both the NTS and the LC.

Animals↗

PubMed2001

NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction.

In response to a variety of external signals, the fungal pathogen Candida albicans undergoes a transition between ellipsoidal single cells (blastospores) and filaments composed of elongated cells attached end-to-end. Here we identify a DNA-binding protein, Nrg1, that represses filamentous growth in Candida probably by acting through the co-repressor Tup1. nrg1 mutant cells are predominantly filamentous under non-filament-inducing conditions and their colony morphology resembles that of tup1 mutants. We also identify two filament-specific genes, ECE1 and HWP1, whose transcription is repressed by Nrg1 under non-inducing conditions. These genes constitute a subset of those under Tup1 control, providing further evidence that Nrg1 acts by recruiting Tup1 to target genes. We show that growth in serum at 37 degrees C, a potent inducer of filamentous growth, causes a reduction of NRG1 mRNA, suggesting that filamentous growth is induced by the down-regulation of NRG1. Consistent with this idea, expression of NRG1 from a non-regulated promoter partially blocks the induction of filamentous growth.

Amino Acid Sequence↗

PubMed2001

Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad.

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, we cloned a DAZ-like sequence from axolotls, Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells.

Ambystoma↗

PubMed2001

Axbrn-1: a maternal transcript encodes a POU transcription factor that is later expressed in the developing central nervous system of axolotl embryos.

Axbrn-1 encodes a class III POU protein with a POU-specific domain and a POU homeodomain that is most similar to RHS2 from rat. In embryos Axbrn-1 transcripts are maternally inherited and persist through cleavage. New transcripts accumulate beginning at the mid-blastula transition. In gastrulating embryos Axbrn-1 RNA is in the dorsal marginal zone and by neurula it is in the neural folds and neural plate. Later, it is specific to the developing brain and anterior spinal cord. Transcriptional activation at the mid-blastula transition indicates that Axbrn-1 is among the earliest genes to be expressed in axolotl embryos.

Ambystoma mexicanum↗

PubMed2001

Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus.

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.

Ambystoma↗

PubMed2001

Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans.

Candida albicans, the major fungal pathogen in humans, can undergo a reversible transition from ellipsoidal single cells (blastospores) to filaments composed of elongated cells attached end to end. This transition is thought to allow for rapid colonization of host tissues, facilitating the spread of infection. Here, we report the identification of Rfg1, a transcriptional regulator that controls filamentous growth of C. albicans in an environment-dependent manner. Rfg1 is important for virulence of C. albicans in a mouse model and is shown to control a number of genes that have been implicated in this process. The closest relative to Rfg1 in Saccharomyces cerevisiae is Rox1, a key repressor of hypoxic genes. However, Rfg1 does not appear to play a role in the regulation of hypoxic genes in C. albicans. These results demonstrate that a regulatory protein that controls the hypoxic response in S. cerevisiae controls filamentous growth and virulence in C. albicans. The observations described in this paper raise new and intriguing questions about the evolutionary relationship between these processes.

Amino Acid Sequence↗

PubMed2001

EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development.

The Pax gene egl-38 plays an important role in the development of several organs in C. elegans. To understand how a Pax transcription factor influences distinct developmental choices in different cells and tissue types, we have characterized a second gene, lin-48. lin-48 functions with egl-38 in the development of one structure, the hindgut, but not in other tissues such as the egg-laying system. We show that lin-48 encodes a C2H2 zinc-finger protein that is similar to the product of the Drosophila gene ovo and is expressed in the hindgut cells that develop abnormally in lin-48 mutants. We present evidence that lin-48 is a target for EGL-38 in hindgut cells. We show that lin-48 requires egl-38 for its expression in the hindgut. Using deletion analysis, we have identified two elements in the lin-48 promoter that are necessary for lin-48 expression. We demonstrate that EGL-38 binds with high affinity to one of these elements. In addition, we have observed genetic interactions between mutations in the lin-48 promoter and specific alleles of egl-38. These experiments demonstrate a functional link between Pax and Ovo transcription factors, and provide a model for how Pax transcription factors can regulate different target genes in different cells.

Alleles↗

PubMed2000

Intravenous nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure. Nesiritide Study Group.

BACKGROUND: Intravenous infusion of nesiritide, a brain (B-type) natriuretic peptide, has beneficial hemodynamic effects in patients with decompensated congestive heart failure. We investigated the clinical use of nesiritide in such patients. METHODS: Patients hospitalized because of symptomatic congestive heart failure were enrolled in either an efficacy trial or a comparative trial. In the efficacy trial, which required the placement of a Swan-Ganz catheter, 127 patients with a pulmonary-capillary wedge pressure of 18 mm Hg or higher and a cardiac index of 2.7 liters per minute per square meter of body-surface area or less were randomly assigned to double-blind treatment with placebo or nesiritide (infused at a rate of 0.015 or 0.030 microg per kilogram of body weight per minute) for six hours. In the comparative trial, which did not require hemodynamic monitoring, 305 patients were randomly assigned to open-label therapy with standard agents or nesiritide for up to seven days. RESULTS: In the efficacy trial, at six hours, nesiritide infusion at rates of 0.015 and 0.030 microg per kilogram per minute decreased pulmonary-capillary wedge pressure by 6.0 and 9.6 mm Hg, respectively (as compared with an increase of 2.0 mm Hg with placebo, P<0.001), resulted in improvements in global clinical status in 60 percent and 67 percent of the patients (as compared with 14 percent of those receiving placebo, P<0.001), reduced dyspnea in 57 percent and 53 percent of the patients (as compared with 12 percent of those receiving placebo, P<0.001), and reduced fatigue in 32 percent and 38 percent of the patients (as compared with 5 percent of those receiving placebo, P<0.001). In the comparative trial, the improvements in global clinical status, dyspnea, and fatigue were sustained with nesiritide therapy for up to seven days and were similar to those observed with standard intravenous therapy for heart failure. The most common side effect was dose-related hypotension, which was usually asymptomatic. CONCLUSIONS: In patients hospitalized with decompensated congestive heart failure, nesiritide improves hemodynamic function and clinical status. Nesiritide is useful for the treatment of decompensated congestive heart failure.

Atrial Natriuretic Factor↗

PubMed2000

Evidence for mating of the "asexual" yeast Candida albicans in a mammalian host.

Since its classification nearly 80 years ago, the human pathogen Candida albicans has been designated as an asexual yeast. In this report, we describe the construction of C. albicans strains that were subtly altered at the mating-type-like (MTL) locus, a cluster of genes that resembles the mating-type loci of other fungi. These derivatives were capable of mating after inoculation into a mammalian host. C. albicans is a diploid organism, but most of the mating products isolated from a mouse host were tetrasomic for the two chromosomes that could be rigorously monitored and, overall, exhibited substantially higher than 2n DNA content. These observations demonstrated that C. albicans can recombine sexually.

Animals↗

PubMed2000

Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast.

The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.

Amino Acid Sequence↗

PubMed2000

The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor.

CHD proteins are members of the chromo domain family, a class of proteins involved in transcription, DNA degradation and chromatin structure. In higher eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4. Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate transcription via alteration of chromatin structure. However, little is known about the role of CHD proteins in vivo, particularly the CHD1 subfamily. To understand better the cellular function of CHD proteins, we initiated a study on the Chd1p protein from budding yeast. Using genomic DNA arrays, we identified genes whose expression is affected by the absence of Chd1p. A synthetic-lethal screen uncovered genetic interactions between SWI/SNF genes and CHD1. Biochemical experiments using Chd1p purified from yeast showed that it reconfigures the structure of nucleosome core particles in a manner distinct from the SWI-SNF complex. Taken together, these results suggest that Chd1p functions as a nucleosome remodeling factor, and that Chd1p may share overlapping roles with the SWI-SNF complex to regulate transcription.

Adenosine Triphosphate↗

PubMed2000

A sequence resembling a peroxisomal targeting sequence directs the interaction between the tetratricopeptide repeats of Ssn6 and the homeodomain of alpha 2.

The tetratricopeptide repeat (TPR) is a 34-aa sequence motif, typically found in tandem clusters, that occurs in proteins of bacteria, archea, and eukaryotes. TPRs interact with other proteins, although few details on TPR-protein interactions are known. In this paper we show that a portion of a loop in the homeodomain of the DNA-binding protein alpha2 is required for its recognition by the TPRs of the corepressor Ssn6. The amino acid sequence of this loop is similar to the sequences recognized by the TPRs of an entirely different protein, Pex5, which directs peroxisomal import. We further show that alpha2 can be made to bind specifically in vitro to the TPRs of Pex5 and that a point mutation that disrupts the alpha2-Ssn6 interaction also disrupts the alpha2-Pex5 interaction. These results demonstrate that two different TPR proteins recognize their target by a similar mechanism, raising the possibility that other TPR-target interactions could occur through the same means.

Base Sequence↗

PubMed2000

Turning genes off by Ssn6-Tup1: a conserved system of transcriptional repression in eukaryotes.

The Ssn6-Tup1 repressor forms one of the largest and most important gene-regulatory circuits in budding yeast. This circuit, which appears conserved in flies, worms and mammals, exemplifies how a 'global' repressor (i.e. a repressor that regulates many genes in the cell) can be highly selective in the genes it represses. It also explains how, given the appropriate signal, specific subsets of these genes can be derepressed. Ssn6-Tup1 seems especially robust, bringing about a high level of repression irrespective of its precise placement on DNA or of specific features of the DNA control regions of its target genes. This high degree of repression probably results from several distinct mechanisms acting together.

Chromatin↗

PubMed2000

Isolation and characterization of myogenic satellite cells from the muscular dystrophic hamster.

Myogenic satellite cells were isolated from control and dystrophic hamster diaphragms to examine cellular mechanisms involved in the physiology of muscular dystrophy. The Bio 14.6 dystrophic hamster, which possesses a defect in the delta-sarcoglycan gene, develops biochemical and physical symptoms of Duchenne-like and limb girdle muscular dystrophies. Because primary cultures of the control and dystrophic satellite cells became extensively contaminated with non-myogenic cells during proliferation, cell clones were developed to provide pure cultures for study. Cell culture conditions were optimized with the use of Ham's F-12K medium containing 10% fetal bovine serum +5% horse serum + 10 ng/mL basic fibroblast growth factor + 50 microg/mL porcine gelatin. Proliferation rates of the two clonal cultures were similar between the two lines. Satellite cell-derived myotubes from both primary cultures and clones differed between control and dystrophic animals. Dystrophic myotubes tended to be long and narrow, while the control-derived myotubes were broader. Measurement of muscle-specific creatine kinase during differentiation revealed that the dystrophic myotubes possessed higher creatine kinase levels than control myotubes (up to 146-fold at 168 h). The results demonstrate that satellite cells can be isolated from the hamster and may provide a useful tool to study muscular dystrophies associated with defects in the sarcoglycan complex and the involvement of sarcoglycans in normal skeletal muscle growth and development.

Animals↗

PubMed2000

TUP1, CPH1 and EFG1 make independent contributions to filamentation in candida albicans.

The common fungal pathogen, Candida albicans, can grow either as single cells or as filaments (hyphae), depending on environmental conditions. Several transcriptional regulators have been identified as having key roles in controlling filamentous growth, including the products of the TUP1, CPH1, and EFG1 genes. We show, through a set of single, double, and triple mutants, that these genes act in an additive fashion to control filamentous growth, suggesting that each gene represents a separate pathway of control. We also show that environmentally induced filamentous growth can occur even in the absence of all three of these genes, providing evidence for a fourth regulatory pathway. Expression of a collection of structural genes associated with filamentous growth, including HYR1, ECE1, HWP1, ALS1, and CHS2, was monitored in strains lacking each combination of TUP1, EFG1, and CPH1. Different patterns of expression were observed among these target genes, supporting the hypothesis that these three regulatory proteins engage in a network of individual connections to downstream genes and arguing against a model whereby the target genes are regulated through a central filamentous growth pathway. The results suggest the existence of several distinct types of filamentous forms of C. albicans, each dependent on a particular set of environmental conditions and each expressing a unique set of surface proteins.

Candida albicans↗

PubMed2000

Identification and characterization of TUP1-regulated genes in Candida albicans.

TUP1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans. Using subtractive hybridization, we identified six genes, termed repressed by TUP1 (RBT), whose expression is regulated by TUP1. One of the genes (HWP1) has previously been characterized, and a seventh TUP1-repressed gene (WAP1) was recovered due to its high similarity to RBT5. These genes all encode secreted or cell surface proteins, and four out of the seven (HWP1, RBT1, RBT5, and WAP1) encode putatively GPI-modified cell wall proteins. The remaining three, RBT2, RBT4, and RBT7, encode, respectively, an apparent ferric reductase, a plant pathogenesis-related protein (PR-1), and a putative secreted RNase T2. The expression of RBT1, RBT4, RBT5, HWP1, and WAP1 was induced in wild-type cells during the switch from the yeast form to filamentous growth, indicating the importance of TUP1 in regulating this process and implicating the RBTs in hyphal-specific functions. We produced knockout strains in C. albicans for RBT1, RBT2, RBT4, RBT5, and WAP1 and detected no phenotypes on several laboratory media. However, two animal models for C. albicans infection, a rabbit cornea model and a mouse systemic infection model, revealed that rbt1Delta and rbt4Delta strains had significantly reduced virulence. TUP1 appears, therefore, to regulate many genes in C. albicans, a significant fraction of which are induced during filamentous growth, and some of which participate in pathogenesis.

Amino Acid Sequence↗

PubMed2000

Expression of the cardiac actin gene in axolotl embryos.

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.

Actins↗