Sall4 regulates downstream patterning genes during limb regeneration.

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

Genetic and molecular characterization of GAL83: its interaction and similarities with other genes involved in glucose repression in Saccharomyces cerevisiae.

Expression of the GAL genes of Saccharomyces cerevisiae is subject to glucose repression, a global regulatory mechanism that requires several gene products. We have isolated GAL83, one of these genes required for glucose repression. The sequence of the predicted Gal83 protein is homologous to two other yeast proteins, Sip1p and Sip2p, which are known to interact with the SNF1 gene product, a protein kinase required for expression of the GAL genes. High-copy clones of SIP1 and SIP2 cross-complement the GAL83-2000 mutation (as well as GAL82-1, a mutation in another gene involved in glucose repression), suggesting that these four genes may perform similar functions in glucose repression. Consistent with this hypothesis, a gal83 null mutation does not affect glucose repression, and only dominant or partially dominant mutations exist in GAL83 (and GAL82). Two other observations were made that suggests that GAL83 functions interdependently with GAL82 and REG1 (another gene involved in glucose repression) to effect glucose repression: 1) REG1 on a low-copy plasmid cross-complements GAL82-1 and GAL83-2000 mutations, and 2) all pairwise combinations of reg1, GAL82-1 and GAL83-2000 fail to complement one another. Such unlinked noncomplementation suggests that Gal83p, Gal82p and Reg1p may interact with one another. Possible roles for GAL83, GAL82 and REG1 are discussed in relation to SNF1, SIP1 and SIP2.

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.

Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo.

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Escherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura+ transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.

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.

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.

Cocaine immunotoxicity: abnormal cytokine production in Hispanic drug users.

Peripheral blood lymphocytes from 47 Hispanic poly-drug users with a history of cocaine abuse were analyzed for in vitro production of interleukin-1 (IL-1), interleukin-2 (IL-2), gamma-interferon (IFN) and plasma levels of soluble IL-2 receptor (SIL-2R). Cocaine use was confirmed and quantified by analysis of hair and urine samples, and subjects were grouped into 3 based on the extent of cocaine metabolites detected. No significant differences in IL-1 and IFN production were seen between the 3 groups. However, subjects with higher levels of cocaine in hair also showed higher levels of IL-2. In addition, a positive correlation was seen between cocaine concentrations and IL-2 levels. A corresponding negative correlation was seen between cocaine levels and levels of plasma SIL-2R. These findings suggest modulation of the IL-2 network by cocaine in poly-drug users.

Immunotoxicity of poly-drug use: abnormalities in the active and high-affinity CD2 antigen (E-rosette receptor) bearing T-lymphocytes.

The E-rosetting profiles of T-cells were studied in 47 subjects with a history of poly-drug and alcohol abuse, and compared with 15 normal controls. No change was evident in numbers of total rosette-forming cells (TRFC). However, there was reduction in active and high-affinity rosette-forming cells (ARFC and HARFC). These two subsets of CD2-antigen-bearing T-cells are considered as immunocompetent surveillance cells. Thus the abnormality associated with them could be due to the combined immunotoxic effects of substance abuse, modulating the immune status of drug users.

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.

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.

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.

Comparison of rural and urban certified nurse-midwives in Arizona.

A survey of certified nurse-midwives (CNMs) in Arizona was carried out in 1990 to provide data for maternity service planning in the state. Information was gathered on location and scope of CNM practice, barriers to practice, and the contribution of CNMs to maternity care. Demographic and clinical practice characteristics of urban and rural CNMs were also compared. Urban and rural CNMs are significantly different in terms of education (urban CNMs are much more likely to have master's degrees) and number of years since first certification (urban CNMs have been certified significantly longer). Rural midwives are more likely to be under the age of 40. Health services provided by urban and rural CNMs were compared with each other and with national data. Midwives in rural areas of Arizona are more likely to provide comprehensive nurse-midwifery services than are either urban Arizona midwives or U.S. midwives as a whole. Urban and rural CNMs described lack of physician backup as a major barrier to nurse-midwifery practice in rural areas. Lack of hospital privileges was another major obstacle noted by rural nurse-midwives. Arizona CNMs felt they could provide comprehensive, cost-effective maternity services in rural areas that would improve access to care, patient satisfaction, and maternal and child health outcomes.

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.

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.

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.

Case management--a bottom-line care delivery model. Part II: Adaptation of the model.

Tuscson Medical Center's service volume management, research based approach to developing an adaptation of several design components of the New England Medical Center's case management model is described. The concept behind this model has been described (JONA 19(11):16-20). TMC's adaptation has been accomplished by operationalizing Rhea's steps to control variation in resource use through the (1) application of qualitative and quantitative research methods, (2) integration of other models, (3) inherent differences between the southwest and northeast settings, and (4) an organizational and participative planning structure and process.