Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells.

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.

Neurovesical dysfunction in postural tachycardia syndrome (POTS).

This is the first report of neurovesical dysfunction in a woman with postural tachycardia syndrome (POTS). The patient had both symptoms and urodynamic findings diagnostic of detrusor hyperreflexia. Management consisted of anticholinergic medication and timed voiding. Lower urinary tract dysfunction may be underrecognized in POTS.

Growth and development in term infants fed long-chain polyunsaturated fatty acids: a double-masked, randomized, parallel, prospective, multivariate study.

OBJECTIVE: To evaluate the effects of dietary intake of the long-chain polyunsaturated fatty acids, arachidonic acid (AA), and docosahexaenoic acid (DHA) on multiple indices of infant growth and development. DESIGN: A double-masked, randomized, parallel trial was conducted with term infants fed formulas with or without AA+DHA for 1 year (N = 239). Reference groups of breastfed infants (N = 165) weaned to formulas with and without AA+DHA were also studied. Infants in the formula groups were randomized at

Lysophosphatidic acid prevents apoptosis in fibroblasts via G(i)-protein-mediated activation of mitogen-activated protein kinase.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with multiple biological functions. In the present study, we demonstrate that, besides its mitogenic activity, LPA is a potent survival factor, preventing serum-deprivation-induced apoptosis in fibroblasts and other cell types. Both the proliferative effect and survival activity of LPA are sensitive to the action of pertussis toxin (PTX), indicating that both processes are mediated by G(i) protein(s). We therefore focused on the role of G(i)-protein-mediated signalling events in the promotion of cell survival by LPA. In addition to activation of mitogen-activated protein kinase (MAPK), LPA stimulates a modest PTX-sensitive phosphorylation/activation of the serine/threonine kinase Akt, a survival mediator downstream of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K with LY 294002 or wortmannin resulted in a marked inhibition of LPA-induced DNA synthesis, and yet the survival activity of LPA decreased by only 20-30%, suggesting a limited input of the PI3K-Akt cascade in LPA-induced cell survival. In contrast, inhibition of MAPK activation by the MEK-1 inhibitor, PD 98059, blocked both the proliferative and survival effects of LPA. These results indicate that LPA promotes cell survival largely via G(i)-protein-mediated activation of ERK1/ERK2, or other PD 98059-sensitive member(s) of the MAPK family.

Lysophospholipid growth factors in the initiation, progression, metastases, and management of ovarian cancer.

Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein-coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein-coupled family of receptors, making the LPA receptor family a "drugable" target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Tolterodine for overactive bladder: time to onset of action, preferred dosage, and 9-month follow-up.

This is a prospective study of 28 patients who had urinary frequency (>8 times/day) and either urgency or urge incontinence (>1 time/day). After a 2-week run-in period (visit 1), the patients were started on tolterodine 1 mg twice a day (bid) (visit 2). They were followed at 4 and 8 weeks (visits 3 and 4). The patients were contacted by telephone 1 week after visit 2. Tolterodine was increased to 2 mg bid if the patient had incomplete improvement at either the initial phone call or during visit 3. Evaluation criteria were daily micturition charts including urinary frequency, nocturia, leakage episodes, average urine volume per day, and average voided volume. Tolterodine was well tolerated without side effects in 20 (80%) of 28 patients. Eight patients (20%) dropped out after enrollment because of side effects in 3, no improvement in 2, and missing visits (>1) in 3. Drug dosage in the 20 patients who tolerated tolterodine was 1 mg bid in 3 and 2 mg bid in 17 (85%). According to micturition charts, urinary frequency, nocturia, and leakage episodes decreased significantly after tolterodine treatment, whereas average urine volume per day and average voided volume did not change significantly. There were no electrocardiographic or biochemical abnormalities due to tolterodine treatment. Mean follow-up was 9.4 months. All 20 patients who tolerated tolterodine continue to take the medication without significant side effects. We conclude that tolterodine is well tolerated and effective for overactive bladders. Two milligrams bid is the dosage preferred by the majority of patients and the onset of action is seen within 1 week of treatment. Long-term compliance and efficacy are excellent, with no dropout in >9 months of follow-up.

C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells.

Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction.

Naturally occurring analogs of lysophosphatidic acid elicit different cellular responses through selective activation of multiple receptor subtypes.

Lysophosphatidic acid (LPA), plasmalogen-glycerophosphate (alkenyl-GP) and, cyclic-phosphatidic acid (cyclic-PA) are naturally occurring phospholipid growth factors (PLGFs). PLGFs elicit diverse biological effects via the activation of G protein-coupled receptors in a variety of cell types. In NIH3T3 fibroblasts, LPA and alkenyl-GP both induced proliferation, whereas cyclic-PA was antiproliferative. LPA and alkenyl-GP decreased cAMP in a pertussis toxin-sensitive manner, whereas cyclic-PA caused cAMP to increase. LPA and alkenyl-GP both stimulated the activity of the mitogen-actived protein kinases extracellular signal regulated kinases 1 and 2 and c-Jun NH2-terminal kinase, whereas cyclic-PA did not. All three PLGFs induced the formation of stress fibers in NIH3T3 fibroblasts. To determine whether these lipids activated the same or different receptors, heterologous desensitization patterns were established among the three PLGFs by monitoring changes in intracellular Ca2+ in NIH3T3 fibroblasts. LPA cross-desensitized both the alkenyl-GP and cyclic-PA responses. Alkenyl-GP cross-desensitized the cyclic-PA response, but only partially desensitized the LPA response. Cyclic-PA only partially desensitized both the alkenyl-GP and LPA responses. We propose that pharmacologically distinct subsets of PLGF receptors exist that distinguish between cyclic-PA and alkenyl-GP, but are all activated by LPA. We provide evidence that the PSP24 receptor is selective for LPA and not activated by the other two PLGFs. RT-PCR and Northern blot analysis indicate the co-expression of mRNAs encoding the EDG-2, EDG-4, and PSP24 receptors in a variety of cell lines and tissues. However, the lack of mRNA expression for these three receptors in the LPA-responsive Rat-1 and Sp2-O-Ag14 cells suggests that a number of PLGF receptor subtypes remain unidentified.

Identification of a novel growth factor-like lipid, 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) that is present in commercial sphingolipid preparations.

Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1H and 13C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 microM was only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065-C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.

Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid.

We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor. LPA activated the pheromone response pathway in S. cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene. LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product). This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway. The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ. LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation. These results indicate that Edg-2 encodes a highly specific LPA receptor.

Modulation of the immune system by human milk and infant formula containing nucleotides.

OBJECTIVE: To determine whether human milk and nucleotides added to infant formula at levels present in human milk enhance development of the immune system during infancy. METHODS: A 12-month, controlled, randomized and blinded, multisite feeding trial was conducted on two infant formulas: iron-fortified, milk-based control formula (Control) or the same formula fortified with nucleotides (Nucleotide). The level (72 mg/L) and ratio of individual nucleotides selected were patterned after those available in human milk. A third group fed human milk exclusively for 2 months and then human milk or Similac with iron until 12 months of age also was studied. Response to immunizations was chosen to assess development of the immune system. Infants followed the immunization schedule recommended by the American Academy of Pediatrics in 1991. OUTCOME VARIABLES: Antibody responses were determined at 6, 7, and 12 months of age to Haemophilus influenzae type b polysaccharide (Hib), to diphtheria and tetanus toxoids, and to oral polio virus (OPV) immunizations. RESULTS: Of 370 full-term, healthy infants enrolled, 311 completed the study (107 Control, 101 Nucleotide, 103 human milk/Similac with iron). Intake, tolerance, and growth of infants were similar in all three groups. Compared with the Control group 1 month after the third immunization (7 months of age), the Nucleotide group had a significantly higher Hib antibody concentration (geometric mean concentrations of 7.24 vs 4.05 micrograms/mL, respectively), and a significantly higher diphtheria antibody concentration (geometric mean of 1.77 vs 1.38 U/mL). The significantly higher Hib antibody response in the Nucleotide group persisted at 12 months. The antibody responses to tetanus and OPV were not enhanced by nucleotide fortification. There also was an effect of breastfeeding on immune response. Infants who breastfed had significantly higher neutralizing antibody titers to polio virus than either formula-fed group (1:346 vs 1:169 and 1:192 in the Control and Nucleotide groups, respectively) at 6 months of age. CONCLUSION: Infant formula fortified with nucleotides enhanced H influenzae type b and diphtheria humoral antibody responses. Feeding human milk enhanced antibody responses to OPV. Dietary factors play a role in the antibody response of infants to immunization.

How race affects health services use by older women.

In this study, we examined the relationships among functional health and its correlates in a sample of 101 low-income, older African American (n = 32), White (n = 37), and Hispanic (n = 32) women residing in the southwestern United States. Statistically significant associations were found among age, education, and income and the functional health variables of physical health, independent health, and psychosocial health. Hispanic women reported higher levels of physical health than White. African American women reported higher levels of both independent and psychosocial health than White women. Through regression analysis, physical health was found to be a significant predictor of the need for and use of health services. Little emphasis has been placed on the functional health needs of older women and race in policy and practice.

Human immunodeficiency virus infection risk among female sex partners of intravenous drug users in southern Arizona.

Epidemiologic data suggest that women who are sexual partners of intravenous drug users (IVDUs) are at increasing risk for infection with human immunodeficiency virus (HIV). The article reports a study describing living conditions, sex risk behaviors, knowledge about acquired immunodeficiency syndrome (AIDS), and perceptions of AIDS risk among female sexual partners of IVDUs living in southern Arizona. One hundred and twenty-three women who did not use IV drugs but had had sex with an IVDU in the last 6 months were interviewed. Eighty percent belonged to an ethnic minority, and 20% were white. Condom use was infrequent regardless of the number of sexual contacts. Sex was primarily heterosexual, with unsafe vaginal intercourse being the most common practice. Barriers to condom use were self-related and partner related. Some women lacked knowledge about sexual transmission of AIDS. All women reported getting AIDS information in the last 6 months and felt some risk of contracting the disease. AIDS risk reduction interventions should include HIV education and focus on barriers to condom use.

Maspin is an intracellular serpin that partitions into secretory vesicles and is present at the cell surface.

The tumor suppressor maspin (mammary serpin) was originally identified as a component of human mammary epithelial cells that is downregulated as mammary tumor cells progress from the benign to the invasive and metastatic states. Maspin inhibits cellular invasion, motility, and proliferation, but its mechanism of action is currently unknown. Because the cellular machinery responsible for these processes is cytoplasmic, we have reexamined the tissue distribution and subcellular localization of maspin. We find that maspin, or a maspin-like protein, is present in many human organs, in which it localizes to epithelia. In cultured human mammary myoepithelial cells, maspin is predominantly a soluble cytoplasmic protein that associates with secretory vesicles and is present at the cell surface. In vitro assays show that the vesicle association is due to the existence of an uncleaved facultative secretion signal that allows small amounts of maspin to partition into the endoplasmic reticulum. These results demonstrate that maspin is more widespread than previously believed. The subcellular localization studies indicate that soluble intracellular and vesicle-associated maspin probably play an important role in controlling the invasion, motility, and proliferation of cells expressing it, whereas extracellular maspin may also regulate these processes in adjacent cells.

Fetal heart rate changes after intrathecal sufentanil or epidural bupivacaine for labor analgesia: incidence and clinical significance.

The objective of this study was to compare the incidence of intrapartum fetal heart tracing (FHT) abnormalities and the obstetric outcome after intrathecal sufentanil (ITS) versus epidural bupivacaine (EB). During the period from April to September 1994, 129 patients who met inclusion criteria were prospectively identified during labor at a single university-affiliated hospital. Inclusion criteria included: singleton, gestational age > or = 36 wk, and cephalic presentation. In the ITS group, epidural anesthesia was not administered before 60 min after ITS. Sixty-five consecutive ITS patients were compared to 64 consecutive EB patients. Each FHT was reviewed independently by two obstetricians blinded to the type of analgesia. The FHT characteristics evaluated included baseline rate, variability, and periodic changes. No differences in the incidence of clinically significant FHT abnormalities (recurrent late decelerations and/or bradycardia) were observed between the two groups (ITS 21.5% versus EB 23.4%). The rates of clinically significant FHT abnormalities in both groups was not different when patients with hypotension and medical complications were excluded (16.9% vs 17.1%). In addition, equal rates of hypotension (18.5% vs 17.2%) were noted between the groups. In both groups there was a significantly higher risk of cesarean section in patients whose previously normal FHT became abnormal postanalgesia when compared to patients without a new onset FHT abnormality (ITS 28.6% [4/14] versus 2.0% [1/51], P < 0.01; EB 33.3% [5/15] versus 8.2% [4/49], P < 0.05). This increased risk was associated with an increase in cesarean section for nonreassuring FHT in both groups (ITS 14.3% [2/14] versus 0% [0/51], P = 0.04; EB 13.3% [2/15] versus 0% [0/49], P = 0.05). These results support the conclusion that the incidence of clinically significant FHT abnormalities and hypotension is equivalent in patients receiving ITS when compared to EB within the first hour of administration. During this period, patients should have continuous FHT monitoring since a new onset FHT abnormality unveils and alerts the physicians to a possible compromised fetal condition and a corresponding increased risk of cesarean section.

Willingness for treatment as a predictor of retention and outcomes.

Retention in drug treatment is important to successful outcomes. The purpose of this study was to test assumptions made in the development and implementation of the ASSET project. The three assumptions were that living conditions of the homeless adult drug user influence willingness for treatment; willingness relates to treatment tenure; and, conditions, willingness and time in treatment influence treatment outcomes. Data on alcohol use, drug use, employment and housing as well as motivation, readiness and suitability of treatment were collected from 494 homeless adults at baseline and at follow-up. Data were subjected to multivariate causal analysis using factor analytic structural equations modeling. Practical fit indices were acceptable. The measurement model confirmed a higher order construct labelled willingness encompassing motivation, readiness and suitability. The structural model demonstrated that willingness positively related to treatment tenure; willingness positively influenced change in drug use and housing; and, tenure related positively to change in housing.

Suppressors reveal two classes of glucose repression genes in the yeast Saccharomyces cerevisiae.

We selected and analyzed extragenic suppressors of mutations in four genes--GRR1, REG1, GAL82 and GAL83-required for glucose repression of the GAL genes in the yeast Saccharomyces cerevisiae. The suppressors restore normal or nearly normal glucose repression of GAL1 expression in these glucose repression mutants. Tests of the ability of each suppressor to cross-suppress mutations in the other glucose repression genes revealed two groups of mutually cross-suppressed genes: (1) REG1, GAL82 and GAL83 and (2) GRR1. Mutations of a single gene, SRG1, were found as suppressors of reg1, GAL83-2000 and GAL82-1, suggesting that these three gene products act at a similar point in the glucose repression pathway. Mutations in SRG1 do not cross-suppress grr1 or hxk2 mutations. Conversely, suppressors of grr1 (rgt1) do not cross-suppress any other glucose repression mutation tested. These results, together with what was previously known about these genes, lead us to propose a model for glucose repression in which Grr1p acts early in the glucose repression pathway, perhaps affecting the generation of the signal for glucose repression. We suggest that Reg1p, Gal82p and Gal83p act after the step(s) executed by Grr1p, possibly transmitting the signal for repression to the Snf1p protein kinase.