bluuebunny/crossref_metadata_2025 huggingface dataset:
THE ROLE OF SOCIAL GRANTS IN MITIGATING THE SOCIO‐ECONOMIC IMPACT OF HIV/AIDS IN TWO FREE STATE COMMUNITIES1
Authors: BOOYSEN FRIKKIE , VAN DER BERG SERVAAS
Published: January 2006

Social grants may play an important role in mitigating the impact of HIV/AIDS. Eligibility for these grants is driven in part by the increasing burden of chronic illness, the mounting orphan crisis and the impoverishment of households associated with the epidemic. This article investigates the role of social grants in mitigating the socio‐economic impact of HIV/AIDS in South Africa, using data from a panel study on the household impact of the epidemic. Social grants reduce inequality and decrease the prevalence, depth and severity of poverty in affected households. However, these transfers also have disincentive effects on employment, while non‐uptake is in some cases higher amongst the poorest.
EDUCATION OUTCOMES AND HOUSEHOLD ILLNESS AND DEATH IN THE SOUTH AFRICAN SCHOOL SETTING
Authors: SCHIERHOUT G.H.
Published: January 2006

It is widely recognized that the HIV/AIDS epidemic may significantly disadvantage children's education in severely affected countries, but empirical quantitative data demonstrating effects and children at risk are scant. The main objective of this study was to investigate associations between chronic illness and death in the household and select educational outcomes in a school setting. This study provides information on the extent of household illness and death in schools in two provinces in South Africa and explores the relationship between these events and select educational measures. Based on the study findings, we present some of the challenges facing the education sector with regards to managing and monitoring the impacts of illness and death, including HIV and AIDS on education at school level.
WILL SMALL AND MEDIUM ENTERPRISES PROVIDE HIV/AIDS SERVICES TO EMPLOYEES? AN ANALYSIS OF MARKET DEMAND
Authors: CONNELLY P. , ROSEN S.
Published: January 2006

Background: Although many large businesses have begun offering HIV/AIDS prevention and treatment services to employees, the vast majority of small and medium sized enterprises (SMEs) have not. SMEs face constraints reducing their demand for services. This study identifies and evaluates those constraints to determine the extent to which SMEs can be expected to implement HIV/AIDS programmes, and to identify opportunities for strengthening the role of SMEs in South Africa's response to the epidemic.

Methods: Structured interviews were conducted with a random sample of 80 SMEs located in KwaZulu Natal (KZN) and Gauteng provinces.

Results: About one quarter of the companies sampled provided some HIV/AIDS services to employees and fewer than half incurred any direct costs to provide those services. Although 52 per cent of the companies believed they had lost employees to AIDS, few of those employees were regarded as critical to operations. AIDS accounted for 10 per cent of the overall annual employee turnover of 13 per cent. Few companies incur direct costs in recruiting or training, and just 30 per cent of permanent employees have access to employer sponsored healthcare. HIV/AIDS ranked 9th of 10 major business concerns faced by SMEs.

Conclusions: Since managers believe few employees are leaving the workforce due to HIV/AIDS and SMEs appear to incur few costs to replace workers, managers are relatively unconcerned about HIV/AIDS. Serious demand‐side barriers exist in the market for HIV/AIDS services for SMEs. For most of the SMEs in our survey, the constraints are too great to expect SMEs to play a major role in the national response to AIDS without assistance.
Cryptococcus neoformans
Senses CO
2
through the Carbonic Anhydrase Can2 and the Adenylyl Cyclase Cac1
Authors: Mogensen Estelle Gewiss , Janbon Guilhem , Chaloupka James , Steegborn Clemens , Fu Man Shun , Moyrand Frédérique , Klengel Torsten , Pearson David S. , Geeves Michael A. , Buck Jochen , Levin Lonny R. , Mühlschlegel Fritz A.
Published: January 2006

Cryptococcus neoformans
, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life,
C. neoformans
is confronted with and responds to dramatic variations in CO
2
concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO
2
) to infected hosts (5% CO
2
) is the induction of capsule biosynthesis. In cells, CO
2
is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that
C. neoformans
contains two β-class carbonic anhydrases, Can1 and Can2. We further demonstrate that
CAN2
, but not
CAN1
, is abundantly expressed and essential for the growth of
C. neoformans
in its natural environment, where CO
2
concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during
C. neoformans
growth. Bicarbonate directly activates the
C. neoformans
Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.
The Arf-GTPase-Activating Protein Gcs1p Is Essential for Sporulation and Regulates the Phospholipase D Spo14p
Authors: Connolly Jaime E. , Engebrecht JoAnne
Published: January 2006

SPO14
, encoding the major
Saccharomyces cerevisiae
phospholipase D (PLD), is essential for sporulation and mediates synthesis of the new membrane that encompasses the haploid nuclei that arise through meiotic divisions. PLD catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. PA stimulates Arf-GTPase-activating proteins (Arf-GAPs), which are involved in membrane trafficking events and actin cytoskeletal function. To determine if Spo14p-generated PA mediates its biological response through Arf-GAPs, we analyzed the sporulation efficiencies of cells deleted for each of the five known and potential yeast Arf-GAPs. Only
gcs1Δ
mutants display a sporulation defect similar to that of
spo14
mutants: cells deleted for
GCS1
initiate the sporulation program but are defective in synthesis of the prospore membrane. Endosome-to-vacuole transport is also impaired in
gcs1Δ
cells during sporulation. Furthermore, Arf-GAP catalytic activity, but not the pleckstrin homology domain, is required for both prospore membrane formation and endosome-to-vacuole trafficking. An examination of Gcs1p-green fluorescent protein revealed that it is a soluble protein. Interestingly, cells deleted for
GCS1
have reduced levels of Spo14p-generated PA. Taken together, these results indicate that
GCS1
is essential for sporulation and suggest that
GCS1
positively regulates
SPO14
.
Serum Resistance-Associated Protein Blocks Lysosomal Targeting of Trypanosome Lytic Factor in
Trypanosoma brucei
Authors: Oli Monika W. , Cotlin Laura F. , Shiflett April M. , Hajduk Stephen L.
Published: January 2006

Trypanosoma brucei brucei
is the causative agent of nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I (apoA-I), apolipoprotein L-I (apoL-I), and haptoglobin-related protein (Hpr) provides this innate protection against
T. b. brucei
infection. This HDL subfraction, called trypanosome lytic factor (TLF), kills
T. b. brucei
following receptor binding, endocytosis, and lysosomal localization.
Trypanosoma brucei rhodesiense
, which is morphologically and physiologically indistinguishable from
T. b. brucei
, is resistant to TLF-mediated killing and causes human African sleeping sickness. Human infectivity by
T. b. rhodesiense
correlates with the evolution of a resistance-associated protein (SRA) that is able to ablate TLF killing. To examine the mechanism of TLF resistance, we transfected
T. b. brucei
with an epitope-tagged
SRA
gene. Transfected
T. b. brucei
expressed SRA mRNA at levels comparable to those in
T. b. rhodesiense
and was highly resistant to TLF. In the SRA-transfected cells, intracellular trafficking of TLF was altered, with TLF being mainly localized to a subset of SRA-containing cytoplasmic vesicles but not to the lysosome. These results indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine the organism's susceptibility to TLF.
Candida albicans
Ecm33p Is Important for Normal Cell Wall Architecture and Interactions with Host Cells
Authors: Martinez-Lopez Raquel , Park Hyunsook , Myers Carter L. , Gil Concha , Filler Scott G.
Published: January 2006

Candida albicans ECM33
encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of
C. albicans ecm33
Δ mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of
ECM33
was 50% slower than that of strains containing two intact copies of
ECM33
. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of
ECM33
had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells.
Saccharomyces cerevisiae
expressing
C. albicans ECM33
did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of
ecm33
Δ mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in
C. albicans
.
Pyruvate-Phosphate Dikinase of Oxymonads and Parabasalia and the Evolution of Pyrophosphate-Dependent Glycolysis in Anaerobic Eukaryotes
Authors: Slamovits Claudio H. , Keeling Patrick J.
Published: January 2006

In pyrophosphate-dependent glycolysis, the ATP/ADP-dependent enzymes phosphofructokinase (PFK) and pyruvate kinase are replaced by the pyrophosphate-dependent PFK and pyruvate phosphate dikinase (PPDK), respectively. This variant of glycolysis is widespread among bacteria, but it also occurs in a few parasitic anaerobic eukaryotes such as
Giardia
and
Entamoeba
spp. We sequenced two genes for PPDK from the amitochondriate oxymonad
Streblomastix strix
and found evidence for PPDK in
Trichomonas vaginalis
and other parabasalia, where this enzyme was thought to be absent. The
Streblomastix
and
Giardia
genes may be related to one another, but those of
Entamoeba
and perhaps
Trichomonas
are distinct and more closely related to bacterial homologues. These findings suggest that pyrophosphate-dependent glycolysis is more widespread in eukaryotes than previously thought, enzymes from the pathway coexists with ATP-dependent more often than previously thought and may be spread by lateral transfer of genes for pyrophosphate-dependent enzymes from bacteria.
Cdc42 Is Required for Proper Growth and Development in the Fungal Pathogen
Colletotrichum trifolii
Authors: Chen Changbin , Ha Young-sil , Min Ji-young , Memmott Stephen D. , Dickman Martin B.
Published: January 2006

Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from
Colletotrichum trifolii
(Ct
Cdc42
), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to
Saccharomyces cerevisiae
Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense Ct
Cdc42
or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in
C. trifolii
leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA
Cdc42
mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.
Integration of Transcriptional and Posttranslational Regulation in a Glucose Signal Transduction Pathway in
Saccharomyces cerevisiae
Authors: Kim Jeong-Ho , Brachet Valérie , Moriya Hisao , Johnston Mark
Published: January 2006

Expression of the
HXT
genes encoding glucose transporters in the budding yeast
Saccharomyces cerevisiae
is regulated by two interconnected glucose-signaling pathways: the Snf3/Rgt2-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway. The Snf3 and Rgt2 glucose sensors in the membrane generate a signal in the presence of glucose that inhibits the functions of Std1 and Mth1, paralogous proteins that regulate the function of the Rgt1 transcription factor, which binds to the
HXT
promoters. It is well established that glucose induces degradation of Mth1, but the fate of its paralogue Std1 has been less clear. We present evidence that glucose-induced degradation of Std1 via the SCF
Grr1
ubiquitin-protein ligase and the 26S proteasome is obscured by feedback regulation of
STD1
expression. Disappearance of Std1 in response to glucose is accelerated when glucose induction of
STD1
expression due to feedback regulation by Rgt1 is prevented. The consequence of relieving feedback regulation of
STD1
expression is that reestablishment of repression of
HXT1
expression upon removal of glucose is delayed. In contrast, degradation of Mth1 is reinforced by glucose repression of
MTH1
expression: disappearance of Mth1 is slowed when glucose repression of
MTH1
expression is prevented, and this results in a delay in induction of
HXT3
expression in response to glucose. Thus, the cellular levels of Std1 and Mth1, and, as a consequence, the kinetics of induction and repression of
HXT
gene expression, are closely regulated by interwoven transcriptional and posttranslational controls mediated by two different glucose-sensing pathways.
Cellular and Molecular Biology of
Candida albicans
Estrogen Response
Authors: Cheng Georgina , Yeater Kathleen M. , Hoyer Lois L.
Published: January 2006

Candida albicans
is the most common etiological agent of vaginal candidiasis. Elevated host estrogen levels and the incidence of vaginal candidiasis are positively associated. Elevated estrogen levels may affect host and/or fungal cells. This study investigates the effect of 17-β-estradiol, 17-α-estradiol, ethynyl estradiol, and estriol on several
C. albicans
strains at concentrations ranging from 10
−5
to 10
−10
M. The addition of 17-β-estradiol or ethynyl estradiol to
C. albicans
cells caused an increase in the number of cells forming germ tubes and an increase in germ tube length in a dose- and strain-dependent manner. The addition of 17-α-estradiol or estriol did not have a significant effect on germ tube formation by the cultured cells. Exposure to exogenous estrogens did not significantly change the biomass of any
C. albicans
culture tested. The transcriptional profile of estrogen-treated
C. albicans
cells showed increased expression of
CDR1
and
CDR2
across several strain-estrogen concentration-time point combinations, suggesting that these genes are the most responsive to estrogen exposure. Analysis of strain DSY654, which lacks the
CDR1
and
CDR2
coding sequences, showed a significantly decreased number of germ tube-forming cells in the presence of 17-β-estradiol.
PDR16
was the most highly up-regulated gene in strain DSY654 under these growth conditions. The cell biology and gene expression data from this study led to a model that proposes how components of the phospholipid and sterol metabolic pathways may interact to affect
C. albicans
germ tube formation and length.
SST2, a Regulator of G-Protein Signaling for theCandida albicansMating Response Pathway
Authors: Dignard Daniel , Whiteway Malcolm
Published: January 2006

Candida albicanscontains a functional mating response pathway that is similar to the well-studied system ofSaccharomyces cerevisiae. We have characterized aregulator ofGproteinsignaling (RGS) homolog inC. albicanswith sequence similarity to theSST2gene ofSaccharomyces cerevisiae. Disruption of this gene, which had been designatedSST2, causes an opaqueMTLa/MTLaderivative of strain SC5314 to show hypersensitivity to theC. albicansα-factor. This hypersensitivity generates an enhanced cell cycle arrest detected in halo assays but reduces the overall mating efficiency of the cells. Transcriptional profiling of the pheromone-regulated gene expression in thesst2mutant shows a pattern of gene induction similar to that observed in wild-type cells, but the responsiveness is heightened. This involvement of an RGS in the sensitivity to pheromone is consistent with the prediction that the mating response pathway inC. albicansrequires the activation of a heterotrimeric G protein.
ATP Synthase Is Responsible for Maintaining Mitochondrial Membrane Potential in Bloodstream Form
Trypanosoma brucei
Authors: Brown Silvia V. , Hosking Paul , Li Jinlei , Williams Noreen
Published: January 2006

The mitochondrion of
Trypanosoma brucei
bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F
1
-ATPase α and β subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the β subunit decreased the mRNA and protein levels for the β subunit, as well as the mRNA and protein levels of the α subunit. Similarly, RNAi of α subunit decreased the α subunit transcript and protein levels, as well as the β-subunit transcript and protein levels. In contrast, α and β RNAi knockdown resulted in a 60% increase in the F
0
complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F
0
-32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for α or β subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of α and β RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form
T. brucei
.
Cell Cycle-Dependent Localization and Properties of a Second Mitochondrial DNA Ligase in
Crithidia fasciculata
Authors: Sinha Krishna Murari , Hines Jane C. , Ray Dan S.
Published: January 2006

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kβ) in the trypanosomatid
Crithidia fasciculata
that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kα). LIG kα localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kα. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kα to the kDNA but at a much lower frequency. The mRNA level of LIG kα varies during the cell cycle out of phase with that of
LIG kβ
. LIG kα transcript levels are maximal during the phase when cells contain two nuclei, whereas
LIG kβ
transcript levels are maximal during S phase. The LIG kα protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the
LIG kα
transcript and the instability of the LIG kα protein suggest a possible role of the ligase in regulating minicircle replication.
Multigene Family Encoding 3′,5′-Cyclic-GMP-Dependent Protein Kinases in
Paramecium tetraurelia
Cells
Authors: Kissmehl Roland , Krüger Tim P. , Treptau Tilman , Froissard Marine , Plattner Helmut
Published: January 2006

In the ciliate
Paramecium tetraurelia
, 3′,5′-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current
Paramecium
genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes,
PKG1
-
1
and
PKG2
-
1
, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some
Paramecium
PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca
2+
stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.
Regulation of human male germ cell death by modulators of ATP production
Authors: Erkkila Krista , Kyttanen Sauli , Wikstrom Marten , Taari Kimmo , Hikim Amiya P. Sinha , Swerdloff Ronald S. , Dunkel Leo
Published: January 2006

The understanding of testicular physiology, pathology, and male fertility issues requires knowledge of male germ cell death and energy production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL, activation of caspases-3 and -9, and electron microscopy) by incubating seminiferous tubule segments under hormone- and serum-free conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed apoptosis at 4 h. Uncoupler 2,4-dinitrophenol (DNP) and oligomycin combination also suppressed death at 4 h, as did the DNP alone. Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor further induced apoptosis nor altered the antiapoptotic effects of the mitochondrial inhibitors. Furthermore, Fas system activation did not modify the effects of mitochondrial modulators. After 24 h, delayed male germ cell apoptosis was observed despite the presence of the mitochondrial inhibitors. We conclude that the mitochondrial ATP production machinery plays an important role in regulating in vitro-induced primary pathways of human male germ apoptosis. The ATP synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, the functional electron transport chain, or the membrane potential. We also conclude that there seem to be secondary pathways of human testicular cell apoptosis that do not require mitochondrial ATP production. The present study emphasizes the role of the main catabolic pathways in the complex network of regulating events of male germ cell life and death.
Physical training reverses the increased activity of the hepatic ketone body synthesis pathway in chronically diabetic rats
Authors: El Midaoui Adil , Chiasson Jean Louis , Tancrède Gilles , Nadeau André
Published: January 2006

This study was designed to examine whether the training-induced improvement in the plasma concentration of ketone bodies in experimental diabetes mellitus could be explained by changes in the activity of the hepatic ketone body synthesis pathway and/or the plasma free fatty acid levels. Diabetes mellitus was induced by an intravenous injection of streptozotocin (50 mg/kg), and training was carried out on a treadmill. The plasma concentration of β-hydroxybutyric acid was increased ( P < 0.001) in sedentary diabetic rats, and this was partly reversed by training ( P < 0.001). The plasma concentration of free fatty acids was increased ( P < 0.001) in sedentary diabetic rats, and this was reversed to normal by training ( P < 0.001). Diabetes was also associated with an increased activity of the hepatic ketone body synthesis pathway. When the data are expressed as per total liver, physical training decreased the activity of the hepatic ketone body synthesis pathway by 18% in nondiabetic rats ( P < 0.05) and by 22% in diabetic rats ( P < 0.01), the activity present in trained diabetic rats being not statistically different from that of sedentary control rats. These data suggest that the beneficial effects of physical training on the plasma β-hydroxybutyric acid levels in the diabetic state are probably explained in part by a decrease in the activity of the hepatic ketone body synthesis pathway and in part by a decrease in plasma free fatty acid levels.
Leptin secretion after a high-fat meal in normal-weight rats: strong predictor of long-term body fat accrual on a high-fat diet
Authors: Leibowitz S. F. , Chang G.-Q. , Dourmashkin J. T. , Yun R. , Julien C. , Pamy P. P.
Published: January 2006

The objective of this study was to investigate meal-related endocrine changes that permit one to identify Sprague-Dawley rats at normal weight that are prone (OP) vs. resistant (OR) to obesity. In blood collected via chronic cardiac catheters, a 2-h high-fat meal (HFM, 50% fat, 40 kcal) at dark onset caused a significant increase in leptin, insulin, and triglycerides compared with premeal levels. Similar to patterns in already obese compared with lean rats on a high-fat diet, these meal-induced endocrine changes in normal-weight rats on lab chow were almost twofold larger in OP rats that, compared with OR rats, subsequently accumulated 100% more fat mass on a chronic high-fat diet. These exaggerated endocrine changes were similarly observed in blood collected using a simpler tail vein puncture procedure. In three separate experiments, the HFM-induced rise in leptin was found to be the strongest, positive correlate ( r = +0.58, +0.62 and +0.64) of long-term body fat accrual. The lowest (2–5 ng/ml) vs. highest (6–9 ng/ml) scores for this post-HFM leptin measurement identified distinct OR and OP subgroups, respectively, when they were similar in body weight (340–350 g), premeal leptin (2.6–3.4 ng/ml), and meal size (40 kcal). Subsequent tests in these normal-weight OP rats revealed a distinct characteristic compared with OR rats, namely, exaggerated HFM-induced rise in expression of the orexigenic peptide galanin in the paraventricular nucleus. Thus, with this HFM-induced leptin measurement, OP rats can be identified while still at normal weight and then investigated for mechanisms that contribute to their excessive body fat accrual on a high-fat diet.
Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon
Authors: Dydensborg Anders Bondo , Herring Elizabeth , Auclair Joëlle , Tremblay Eric , Beaulieu Jean-Francois
Published: January 2006

As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include β-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, β-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.
Nongenomic effects of progesterone on the contraction of muscle cells from the guinea pig colon
Authors: Xiao Zuo-Liang , Cao Weibiao , Biancani Piero , Behar Jose
Published: January 2006

Progesterone (PG) affects muscle cells by genomic mechanisms through nuclear receptors and by nongenomic mechanisms through unidentified pathways. This study aimed to determine the pathways mediating its nongenomic actions. Experiments were performed in dissociated muscle cells from guinea pig colons. Nongenomic actions were defined as those occurring within 10 min of PG exposure. PG blocked the contraction to CCK-8 and NKA (10−7M) but did not impair ACh (10−7M) and KCl (2.5 × 10−2M)-induced contraction. Both CCK-8 and NKA contract muscle cells by releasing calcium from intracellular stores, whereas ACh and KCl can utilize extracellular calcium. PG also blocked the contraction induced by inositol 1,4,5-trisphosphate, thapsigargin, and caffeine, agents that contract muscle cells by releasing calcium from storage sites. The nongenomic actions of PG were transient because they were absent 1 h after the first PG dose, remaining unresponsive after a second PG dose was administered. Furthermore, PG had no effect on the contraction induced by CCK-8 and thapsigargin in muscle cells from animals pretreated with daily intramuscular PG for 4 days. Cytosolic incorporation experiments of [3H]PG showed that pretreatment with unlabeled PG significantly reduced the radiolabeled PG incorporation in the cytosol. We conclude that the nongenomic actions of PG on colonic muscle cells transiently blocked calcium release from storage sites, and this response became rapidly desensitized. This effect does not appear to be specific to PG because other steroid hormones such as aldosterone and testosterone can also induce it.
Reactive oxygen species, but not Ca2+overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion
Authors: Kim Jae-Sung , Jin Yingai , Lemasters John J.
Published: January 2006

We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+overloading again occurred late after reperfusion. This late Ca2+overloading was blocked by MPT inhibition. Intramitochondrial Ca2+chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.
Calcium sensitivity of vasospastic basilar artery after experimental subarachnoid hemorrhage
Authors: Macdonald R. Loch , Zhang Zhen-Du , Takahashi Masataka , Nikitina Elena , Young J. , Xie An , Larkin Lisa
Published: January 2006

Arteries that develop vasospasm after subarachnoid hemorrhage (SAH) may have altered contractility and compliance. Whether these changes are due to alterations in the smooth muscle cells or the arterial wall extracellular matrix is unknown. This study elucidated the location of such changes and determined the calcium sensitivity of vasospastic arteries. Dogs were placed under general anesthesia and underwent creation of SAH using the double-hemorrhage model. Vasospasm was assessed by angiography performed before and 4, 7, or 21 days after SAH. Basilar arteries were excised from SAH or control dogs ( n = 8–52 arterial rings from 2–9 dogs per measurement) and studied under isometric tension in vitro before and after permeabilization of smooth muscle with α-toxin. Endothelium was removed from all arteries. Vasospastic arteries demonstrated significantly reduced contractility to KCl with a shift in the EC50toward reduced sensitivity to KCl 4 and 7 days after SAH ( P < 0.05, ANOVA). There was reduced compliance that persisted after permeabilization ( P < 0.05, ANOVA). Calcium sensitivity was decreased during vasospasm 4 and 7 days after SAH, as assessed in permeabilized arteries and in those contracted with BAY K 8644 in the presence of different concentrations of extracellular calcium ( P < 0.05, ANOVA). Depolymerization of actin with cytochalasin D abolished contractions to KCl but failed to alter arterial compliance. In conclusion, it is shown for the first time that calcium sensitivity is decreased during vasospasm after SAH in dogs, suggesting that other mechanisms are involved in maintaining the contraction. Reduced compliance seems to be due to an alteration in the arterial wall extracellullar matrix rather than the smooth muscle cells themselves because it cannot be alleviated by depolymerization of smooth muscle actin.
The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway
Authors: Fetalvero Kristina M. , Shyu Maureen , Nomikos Athena P. , Chiu Yuh-Fang , Wagner Robert J. , Powell Richard J. , Hwa John , Martin Kathleen A.
Published: January 2006

Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM α-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14–22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via Gsactivation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.
Cells expressing unique Na+/Ca2+exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+overload
Authors: Hurtado Cecilia , Prociuk Michele , Maddaford Thane G. , Dibrov Elena , Mesaeli Nasrin , Hryshko Larry V. , Pierce Grant N.
Published: January 2006

The Na+/Ca2+exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+was inhibited by 5 μM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+overload, whereas NCX1.1 promotes Ca2+overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+overload.
Exercise training improves femoral artery blood flow responses to endothelium-dependent dilators in hypercholesterolemic pigs
Authors: Woodman Christopher R. , Ingram David , Bonagura John , Laughlin M. Harold
Published: January 2006

We tested two hypotheses: 1) that the effects of hypercholesterolemia on endothelial function in femoral arteries exceed those reported in brachial arteries and 2) that exercise (Ex) training enhances endothelium-dependent dilation and improves femoral artery blood flow (FABF) in hypercholesterolemic pigs. Adult male pigs were fed a normal fat (NF) or high-fat/cholesterol (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were Ex trained or remained sedentary (Sed) for 16 wk, thus yielding four groups: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. Endothelium-dependent vasodilator responses were assessed in vivo by measuring changes in FABF after intra-arterial injections of ADP and bradykinin (BK). Endothelium-dependent and -independent relaxation was assessed in vitro by measuring relaxation responses to BK and sodium nitroprusside (SNP). FABF increased in response to ADP and BK in all groups. FABF responses to ADP and BK were not impaired by HF but were improved by Ex in HF pigs. BK- and SNP-induced relaxation of femoral artery rings was not altered by HF or Ex. To determine whether the mechanism(s) for vasorelaxation of femoral arteries was altered by HF or Ex, BK-induced relaxation was assessed in vitro in the absence or presence of NG-nitro-l-arginine methyl ester [l-NAME; to inhibit nitric oxide synthase (NOS)], indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. BK-induced relaxation was inhibited by l-NAME and l-NAME + Indo in all groups of femoral arteries. Ex increased the NOS-dependent component of endothelium-dependent relaxation in NF (not HF) arteries. Indo did not inhibit BK-induced relaxation. Collectively, these results indicate that hypercholesterolemia does not alter endothelial function in femoral arteries and that Ex training improves FABF responses to ADP and BK; however, the improvement cannot be attributed to enhanced endothelial function in HF femoral arteries. These data suggest that Ex-induced improvements in FABF in HF arteries are mediated by vascular adaptations in arteries/arterioles downstream from the femoral artery.
Targeted deletion of Puma attenuates cardiomyocyte death and improves cardiac function during ischemia-reperfusion
Authors: Toth Ambrus , Jeffers John R. , Nickson Philip , Min Jiang-Yong , Morgan James P. , Zambetti Gerard P. , Erhardt Peter
Published: January 2006

The p53-upregulated modulator of apoptosis (Puma), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and -independent forms of apoptosis and has been implicated in the pathomechanism of several diseases, including cancer, acquired immunodeficiency syndrome, and ischemic brain disease. The role of Puma in cardiomyocyte death, however, has not been analyzed. On the basis of the ability of Puma to integrate diverse cell death stimuli, we hypothesized that Puma might be critical for cardiomyocyte death upon ischemia-reperfusion (I/R) of the heart. Here we show that hypoxia-reoxygenation of isolated cardiomyocytes led to an increase in Puma mRNA and protein levels. Moreover, if Puma was delivered by an adenoviral construct, cardiomyocytes died by apoptosis. Under ATP-depleted conditions, however, Puma overexpression primarily induced necrosis, suggesting that Puma is involved in the development of both types of cell death. Consistent with these findings, targeted deletion of Puma in a mouse model attenuated both apoptosis and necrosis. When the Langendorff ex vivo I/R model was used, infarcts were ∼50% smaller in Puma−/− than in wild-type mice. As a result, after I/R, cardiac function was significantly better preserved in Puma−/− mice than in their wild-type littermates. Our study thus establishes Puma as an essential mediator of cardiomyocyte death upon I/R injury and offers a novel therapeutic target to limit cell loss in ischemic heart disease.
Hypoxia and hypoxia-inducible factor-1α promote growth factor-induced proliferation of human vascular smooth muscle cells
Authors: Schultz Kelly , Fanburg Barry L. , Beasley Debbie
Published: January 2006

Hypoxia is thought to be a stimulus for the excessive proliferation of vascular smooth muscle cells (VSMC) that contributes to pulmonary hypertension, but the mechanisms involved are unknown. Here we tested whether hypoxia-inducible factor 1-α (HIF-1α), a master regulator of the transcriptional response to hypoxia, is involved in the enhanced mitogen-induced proliferative responses of hypoxic VSMC. Exposure to moderate hypoxia (5% O2) enhanced the proliferative responses of human pulmonary artery SMC (HPASMC) to mitogens including platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF), compared with those in normoxia (20% O2). Moderate hypoxia elicited increased cellular HIF-1α levels, shown by Western blot analysis, and also enhanced PDGF-, FGF-2-, and EGF-induced expression of HIF-1α. Knockdown of HIF-1α or HIF-1β levels in HPASMC with specific small interfering RNAs inhibited FGF-2-stimulated proliferation of HPASMC incubated in either 5% or 20% O2but failed to inhibit the comitogenic effect of hypoxia. Knockdown of HIF-1α similarly inhibited PDGF-stimulated proliferation, whereas HIF-2α knockdown had no effect on HPASMC proliferation. Knockdown of HIF-1α expression also inhibited growth factor-induced expression of cyclin A. We conclude that HIF-1α promotes proliferative responses of human VSMC to FGF-2, PDGF, and EGF by mechanisms that may involve HIF-1-dependent expression of cyclin A, but HIF is apparently not crucial to the enhancement of FGF-2-, PDGF-, and EGF-induced proliferation of VSMC that occurs during hypoxia.