bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Regulation of Ceramide Synthase Components
LAC1
and
LAG1
in
Saccharomyces cerevisiae
Authors: Kolaczkowski Marcin , Kolaczkowska Anna , Gaigg Barbara , Schneiter Roger , Moye-Rowley W. Scott 
Published: August 2004

ABSTRACT

In
Saccharomyces cerevisiae
, the essential ceramide synthase reaction requires the presence of one of a homologous pair of genes,
LAG1
and
LAC1
. Mutants that lack both of these genes cannot produce ceramide and exhibit a striking synthetic growth defect. While the regulation of ceramide production is critical for the control of proliferation and for stress tolerance, little is known of the mechanisms that ensure proper control of this process. The data presented here demonstrate that the pleiotropic drug resistance (Pdr) regulatory pathway regulates the transcription of multiple genes encoding steps in sphingolipid biosynthesis, including
LAC1
. The zinc cluster transcriptional activators Pdr1p and Pdr3p bind to Pdr1p/Pdr3p-responsive elements (PDREs) in the promoters of Pdr pathway target genes.
LAC1
contains a single PDRE in its promoter, but notably,
LAG1
does not. Reporter gene, Northern blot, and Western blot assays indicated that the expression level of Lac1p is approximately three times that of Lag1p. Detailed analyses of the
LAC1
promoter demonstrated that transcription of this gene is inhibited by the presence of the transcription factor Cbf1p and the anaerobic repressor Rox1p.
LAG1
transcription was also elevated in
cbf1
Δ cells, indicating at least one common regulatory input. Although a hyperactive Pdr pathway altered the profile of sphingolipids produced, the loss of either
LAC1
or
LAG1
alone failed to produce further changes. Two other genes involved in sphingolipid biosynthesis (
LCB2
and
SUR2
) were found to contain PDREs in their promoters and to be induced by the Pdr pathway. These data demonstrate extensive coordinate control of sphingolipid biosynthesis and multidrug resistance in yeast.
Role of a 300-Kilodalton Nuclear Complex in the Maturation of
Trypanosoma brucei
Initiator Methionyl-tRNA
Authors: Arhin George K. , Shen Shuiyuan , Irmer Henriette , Ullu Elisabetta , Tschudi Christian 
Published: August 2004

ABSTRACT

tRNAs are transcribed as precursors containing 5′ leader and 3′ extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNA
Met
in
Trypanosoma brucei
, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of
T. brucei
that is required for trimming the 5′ leader of initiator tRNA
Met
precursors. One of the subunits of the complex (
T. brucei
MT40 [TbMT40]) is a putative methyltransferase and a homolog of
Saccharomyces cerevisiae
Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA
Met
containing 5′ extensions but processed 3′ ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA
Met
molecules with 5′ and 3′ extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA
Met
. Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA
Met
, 3′ trimming precedes 5′ processing.
Ectopic Expression of
URA3
Can Influence the Virulence Phenotypes and Proteome of
Candida albicans
but Can Be Overcome by Targeted Reintegration of
URA3
at the
RPS10
Locus
Authors: Brand Alexandra , MacCallum Donna M. , Brown Alistair J. P. , Gow Neil A. R. , Odds Frank C. 
Published: August 2004

ABSTRACT

Uridine auxotrophy, based on disruption of both
URA3
alleles in diploid
Candida albicans
strain SC5314, has been widely used to select gene deletion mutants created in this fungus by “Ura-blasting” and PCR-mediated disruption. We compared wild-type
URA3
expression with levels in mutant strains where
URA3
was positioned either within deleted genes or at the highly expressed
RPS10
locus.
URA3
expression levels differed significantly and correlated with the specific activity of Ura3p, orotidine 5′-monophosphate decarboxylase. Reduced
URA3
expression following integration at the
GCN4
locus was associated with an attenuation of virulence. Furthermore, a comparison of the SC5314 (
URA3
) and CAI-4 (
ura3
) proteomes revealed that inactivation of
URA3
caused significant changes in the levels of 14 other proteins. The protein levels of all except one were partially or fully restored by the reintegration of a single copy of
URA3
at the
RPS10
locus. Transcript levels of genes expressed ectopically at this locus in reconstituted heterozygous mutants also matched the levels found when the genes were expressed at their native loci. Therefore, phenotypic changes in
C. albicans
can be associated with the selectable marker rather than the target gene. Reintegration of
URA3
at an appropriate expression locus such as
RPS10
can offset most problems related to the phenotypic changes associated with gene knockout methodologies.
RSC1
and
RSC2
Are Required for Expression of Mid-Late Sporulation-Specific Genes in
Saccharomyces cerevisiae
Authors: Bungard David , Reed Michelle , Winter Edward 
Published: August 2004

ABSTRACT

Rsc1 and Rsc2 are alternative bromodomain-containing subunits of the ATP-dependent RSC chromatin remodeling complex in
Saccharomyces cerevisiae
. Smk1 is a sporulation-specific mitogen-activated protein kinase homolog that is required for the postmeiotic events of spore formation. In this study we show that
RSC1
and
RSC2
are haploinsufficient for spore formation in a
smk1
hypomorph. Moreover, diploids lacking Rsc1 or Rsc2 show a subset of
smk1
-like phenotypes. High-copy-number
RSC1
plasmids do not suppress
rsc2-
Δ/
rsc2-
Δ sporulation defects, and high-copy-number
RSC2
plasmids do not suppress
rsc1-
Δ/
rsc1-
Δ sporulation defects. Mid-late sporulation-specific genes, which are normally expressed while key steps in spore assembly occur and which include genes that are required for spore wall formation, are not expressed in cells lacking Rsc1 or Rsc2. We speculate that the combined action of Rsc1 and Rsc2 at mid-late promoters is specifically required for the proper expression of this uniquely timed set of genes. Our data suggest that Smk1 and Rsc1/2 define parallel pathways that converge to provide signaling information and the expression of gene products, respectively, that are required for spore morphogenesis.
Gpr1, a Putative G-Protein-Coupled Receptor, Regulates Morphogenesis and Hypha Formation in the Pathogenic Fungus
Candida albicans
Authors: Miwa Takuya , Takagi Yukinobu , Shinozaki Makiko , Yun Cheol-Won , Schell Wiley A. , Perfect John R. , Kumagai Hidehiko , Tamaki Hisanori 
Published: August 2004

ABSTRACT

In response to various extracellular signals, the morphology of the human fungal pathogen
Candida albicans
switches from yeast to hypha form. Here, we report that
GPR1
encoding a putative G-protein-coupled receptor and
GPA2
encoding a Gα subunit are required for hypha formation and morphogenesis in
C. albicans
. Mutants lacking Gpr1 (
gpr1/gpr1
) or Gpa2 (
gpa2/gpa2
) are defective in hypha formation and morphogenesis on solid hypha-inducing media. These phenotypic defects in solid cultures are suppressed by exogenously added dibutyryl-cyclic AMP (dibutyryl-cAMP). Biochemical studies also reveal that
GPR1
and
GPA2
are required for a glucose-dependent increase in cellular cAMP. An epistasis analysis indicates that Gpr1 functions upstream of Gpa2 in the same signaling pathway, and a two-hybrid assay reveals that the carboxyl-terminal tail of Gpr1 interacts with Gpa2. Moreover, expression levels of
HWP1
and
ECE1
, which are cAMP-dependent hypha-specific genes, are reduced in both mutant strains. These findings support a model that Gpr1, as well as Gpa2, regulates hypha formation and morphogenesis in a cAMP-dependent manner. In contrast,
GPR1
and
GPA2
are not required for hypha formation in liquid fetal bovine serum (FBS) medium. Furthermore, the
gpr1
and the
gpa2
mutant strains are fully virulent in a mouse infection. These findings suggest that Gpr1 and Gpa2 are involved in the glucose-sensing machinery that regulates morphogenesis and hypha formation in solid media via a cAMP-dependent mechanism, but they are not required for hypha formation in liquid medium or during invasive candidiasis.
Discovery of Cercosporamide, a Known Antifungal Natural Product, as a Selective Pkc1 Kinase Inhibitor through High-Throughput Screening
Authors: Sussman Andrea , Huss Karen , Chio Li-Chun , Heidler Steve , Shaw Margaret , Ma Doreen , Zhu Guoxin , Campbell Robert M. , Park Tae-Sik , Kulanthaivel Palaniappan , Scott John E. , Carpenter John W. , Strege Mark A. , Belvo Matthew D. , Swartling James R. , Fischl Anthony , Yeh Wu-Kuang , Shih Chuan , Ye Xiang S. 
Published: August 2004

ABSTRACT

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broad-spectrum fungicidal antifungal drugs through a
Candida albicans
Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which
Saccharomyces cerevisiae
cell wall mutants were found to be highly sensitive to cercosporamide. Indeed,
S. cerevisiae
mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a β-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and β-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.
Proteomic Analysis of
Candida albicans
Cell Walls Reveals Covalently Bound Carbohydrate-Active Enzymes and Adhesins
Authors: de Groot Piet W. J. , de Boer Albert D. , Cunningham Jeff , Dekker Henk L. , de Jong Luitzen , Hellingwerf Klaas J. , de Koster Chris , Klis Frans M. 
Published: August 2004

ABSTRACT

Covalently linked cell wall proteins (CWPs) of the dimorphic fungus
Candida albicans
are implicated in virulence. We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells. Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry [LC/MS/MS]) methods. HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively. In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant β-1,3-glucanase. Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods. The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins. Nonsecretory proteins were absent in our cell wall preparations. The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins. In addition, Pga24p shows similarity to the flocculins of baker's yeast. (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions. The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown. These results indicate that a substantial number of the covalently linked CWPs of
C. albicans
are actively involved in cell wall remodeling and expansion and in host-pathogen interactions.
Dehydration-Specific Induction of Hydrophilic Protein Genes in the Anhydrobiotic Nematode
Aphelenchus avenae
Authors: Browne John A. , Dolan Katharine M. , Tyson Trevor , Goyal Kshamata , Tunnacliffe Alan , Burnell Ann M. 
Published: August 2004

ABSTRACT

Some organisms can survive exposure to extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living nematode
Aphelenchus avenae
can be induced to enter the anhydrobiotic state by exposure to a moderate reduction in relative humidity. During this preconditioning period, the nematode accumulates large amounts of the disaccharide trehalose, which is thought to be necessary, but not sufficient, for successful anhydrobiosis. To identify other adaptations that are required for anhydrobiosis, we developed a novel SL1-based mRNA differential display technique to clone genes that are upregulated by dehydration in
A. avenae
. Three such genes,
Aav-lea-1
,
Aav-ahn-1
, and
Aav-glx-1
, encode, respectively, a late embryogenesis abundant (LEA) group 3 protein, a novel protein that we named anhydrin, and the antioxidant enzyme glutaredoxin. Strikingly, the predicted LEA and anhydrin proteins are highly hydrophilic and lack significant secondary structure in the hydrated state. The dehydration-induced upregulation of
Aav-lea-1
and
Aav-ahn-1
was confirmed by Northern hybridization and quantitative PCR experiments. Both genes were also upregulated by an osmotic upshift, but not by cold, heat, or oxidative stress. Experiments to investigate the relationship between mRNA levels and protein expression for these genes are in progress. LEA proteins occur commonly in plants, accumulating during seed maturation and desiccation stress; the presence of a gene encoding an LEA protein in an anhydrobiotic nematode suggests that some mechanisms of coping with water loss are conserved between plants and animals.
Direct Physical and Functional Interaction of the NuA4 Complex Components Yaf9p and Swc4p
Authors: Bittner Claudia B. , Zeisig Deniz T. , Zeisig Bernd B. , Slany Robert K. 
Published: August 2004

ABSTRACT

Saccharomyces cerevisiae
Yaf9p and the mammalian leukemia-associated protein ENL share a high degree of similarity. To investigate the biological function of Yaf9p, this protein was used to search for interacting proteins in a two-hybrid system. Here, we demonstrate that Yaf9p binds directly to Swc4p, the yeast homolog of the mammalian DNA-methyltransferase-associated protein 1. Yaf9p and Swc4p associate through C-terminal domains, and both proteins coprecipitate in vitro in pull-down experiments and in vivo by immunoprecipitation. In living cells, Swc4p is present in a megadalton protein complex that shows a fractionation behavior in gel filtration similar to that of Esa1p, the histone acetyltransferase of the NuA4 complex. Recruitment of Yaf9p to DNA leads to promoter-specific transcriptional activation that can be inhibited by dominant negative Swc4p lacking the Yaf9p binding domain. Interference with Swc4p function also increases sensitivity to the microtubule toxin benomyl, a trait that corresponds to the known phenotype of a
yaf9
−
knockout strain. In summary, the results suggest that Yaf9p and Swc4p form a protein pair that has a role in chromatin modification with possible implications also for the function of their mammalian counterparts.
CHPA, a Cysteine- and Histidine-Rich-Domain-Containing Protein, Contributes to Maintenance of the Diploid State in
Aspergillus nidulans
Authors: Sadanandom Ari , Findlay Kim , Doonan John H. , Schulze-Lefert Paul , Shirasu Ken 
Published: August 2004

ABSTRACT

The alternation of eukaryotic life cycles between haploid and diploid phases is crucial for maintaining genetic diversity. In some organisms, the growth and development of haploid and diploid phases are nearly identical, and one might suppose that all genes required for one phase are likely to be critical for the other phase. Here, we show that targeted disruption of the
chpA
(cysteine- and histidine-rich-domain- [CHORD]-containing protein A) gene in haploid
Aspergillus nidulans
strains gives rise to
chpA
knockout haploids and heterozygous diploids but no
chpA
knockout diploids.
A. nidulans chpA
heterozygous diploids showed impaired conidiophore development and reduced conidiation. Deletion of
chpA
from diploid
A. nidulans
resulted in genome instability and reversion to a haploid state. Thus, our data suggest a vital role for
chpA
in maintenance of the diploid phase in
A. nidulans
. Furthermore, the human
chpA
homolog,
Chp-1
, was able to complement haploinsufficiency in
A. nidulans chpA
heterozygotes, suggesting that the function of CHORD-containing proteins is highly conserved in eukaryotes.
Dynamics of
Toxoplasma gondii
Differentiation
Authors: Dzierszinski Florence , Nishi Manami , Ouko Lillian , Roos David S. 
Published: August 2004

ABSTRACT

Parasite differentiation is commonly associated with transitions between complex life cycle stages and with long-term persistence in the host, and it is therefore critical for pathogenesis. In the protozoan parasite
Toxoplasma gondii
, interconversion between rapidly growing tachyzoites and latent encysted bradyzoites is accompanied by numerous morphological and metabolic adaptations. In order to explore early cell biological events associated with this differentiation process, we have exploited fluorescent reporter proteins targeted to various subcellular locations. Combining these markers with efficient in vitro differentiation and time-lapse video microscopy provides a dynamic view of bradyzoite development in living cultures, demonstrating subcellular reorganization, maintenance of the mitochondrion, and missegregation of the apicoplast. Bradyzoites divide asynchronously, using both endodyogeny and endopolygeny, and are highly motile both within and between host cells. Cysts are able to proliferate without passing through an intermediate tachyzoite stage, via both the migration of free bradyzoites and the fission of bradyzoite cysts, suggesting a mechanism for dissemination during chronic infection.
Repetitive strain injury
Authors: Helliwell P S , Taylor W J 
Published: August 2004

Abstract
Pain in the forearm is relatively common in the community. In the workplace forearm pain is associated with work involving frequent repetition, high forces, and prolonged abnormal postures. Nevertheless, other factors are involved in the presentation and the continuation of the pain. Notable among these factors are psychosocial issues and the workplace environment—the attitude to workers and their welfare, the physical conditions, and design of the job. Primary prevention may be effective but active surveillance is important with early intervention and an active management approach. Physical treatments have not been extensively evaluated. In the established case, management should be multidisciplinary, addressing physical aspects of the job but also addressing the “yellow, blue, and black flags” which should be viewed as obstacles to recovery. For the worker “on sick” a dialogue should be established between the worker, the primary care physician, and the workplace. Return to work should be encouraged and facilitated by medical interventions and light duty options. Rehabilitation programmes may be of use in chronic cases.
Long term motor complications of levodopa: clinical features, mechanisms, and management strategies
Authors: Thanvi B R , Lo T C N 
Published: August 2004

Abstract
Levodopa is the most effective symptomatic treatment of Parkinson’s disease. However, after an initial period of dramatic benefit, several limitations become apparent including, “dopa resistant” motor symptoms (postural abnormalities, freezing episodes, speech impairment), “dopa resistant” non-motor signs (autonomic dysfunction, mood and cognitive impairment, etc), and/or drug related side effects (especially psychosis, motor fluctuations, and dyskinesias). Motor complications include fluctuations, dyskinesias, and dystonias. They can be very disabling and difficult to treat. Therefore, strategies should ideally be developed to prevent them. Though mechanisms underlying motor complications are only partially understood, recent work has revealed the importance of pulsatile stimulation of postsynaptic dopamine receptors and the disease severity. As a result of intermittent stimulation there occurs a cascade of changes in cell signalling leading to upregulation of the N-methyl-D-aspartate subtype of gamma-aminobutryric acid-ergic neurones. Modified preparations of levodopa (controlled release preparations, liquid levodopa), catecholamine-o-methyltransferase inhibitors, dopamine agonists, amantidine, and various neurosurgical approaches have been used in the prevention and/or treatment of motor complications. Current management of motor complications is less than satisfactory. With better understanding of the pathogenetic mechanisms, it is hoped that future therapeutic strategies will provide a safer and targeted treatment.
Probiotics and prebiotics in the elderly
Authors: Hamilton-Miller J M T 
Published: August 2004

Abstract
Probiotics (usually lactobacilli and bifidobacteria) and prebiotics (non-digestible oligosaccharides) have been shown to be useful in preventing certain disease conditions as well as possibly promoting specific aspects of health. In the present review, the evidence from clinical trials for benefits from probiotics and prebiotics to elderly populations is presented and discussed, specifically in respect of three common conditions found in the elderly. Both probiotics and prebiotics may be helpful in malnutrition, particularly in lactose intolerance and calcium absorption, and in constipation. Probiotics have been shown clearly to boost immunity in the elderly, but the clinical significance of this remains to be clarified. These results are encouraging, and further large scale studies seem justified to establish the place of probiotic and prebiotic supplements in elderly subjects.
Observation and identification of lactate dehydrogenase anomaly in a postburn patient
Authors: Liu Z-J , Zhang Y , Zhang X-B , Yang X 
Published: August 2004

Abstract
Objective: Lactate dehydrogenase (LDH) anomaly is one of the macroenzymes. Macroenzymes are enzymes in serum that have formed high molecular mass complexes, either by self polymerisation or by association with other serum components. The aim of this study was to identify the properties of LDH anomaly and observe the changes from admission to discharge in a postburn patient with LDH anomaly in his serum.
Methods: LDH isoenzymes of the serum were electrophoretically fractionated with terylene cellulose acetate supporting media; LDH anomaly was identified by counter immunoelectrophoresis.
Results: An abnormal LDH-4 band and an extra band on the cathode of LDH-5 were observed in the serum of this patient and were found to be part of an LDH-IgG complex. As his symptoms improved, the patient’s LDH anomaly gradually disappeared. The appearance and disappearance of the anomaly seemed to be related to the progression of the patient’s burns.
Conclusion: In clinical practice, it is important to keep in mind the possibility of an LDH anomaly in patients when the LDH level is abnormally high or does not seem to be related to the clinical state. Early discovery of an LDH anomaly in a patient’s serum may be useful for diagnosis and treatment.