Whole-rat protein content estimation: applicability of the Nx6.25 factor

The amino acid composition of the protein from three strains of rat (Wistar, Zucker lean and Zucker obese), subjected to reference and high-fat diets has been used to determine the mean empirical formula, molecular weight and N content of whole-rat protein. The combined whole protein of the rat was uniform for the six experimental groups, containing an estimate of 17.3% N and a mean aminoacyl residue molecular weight of 103.7. This suggests that the appropriate protein factor for the calculation of rat protein from its N content should be 5.77 instead of the classical 6.25. In addition, an estimate of the size of the non-protein N mass in the whole rat gave a figure in the range of 5.5 % of all N. The combination of the two calculations gives a protein factor of 5.5 for the conversion of total N into rat protein.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Immunoelectron microscopic localization of transforming growth factor alpha in rat colon

Transforming growth factor alpha (TGF alpha) is a polypeptide, which binds to the epidermal growth factor receptor to carry out its function related to cell proliferation and differentiation. The ultrastructural localisation of TGF alpha was studied in both the proximal and the distal colon. The columnar cells, lining the surface epithelium of the proximal colon, showed a strong immunoreactivity in the polyribosomes and in the interdigitations of the lateral membrane. The columnar cells of the crypts and the goblet cells in both the proximal and the distal colon showed the immunostaining in the cis and trans cisternae of the Golgi apparatus. TGF alpha seems to be processed differently in the surface columnar cells and in the crypt columnar cells and goblet cells. Moreover, it probably has different roles in proliferation and differentiation.

Potential impact of single-risk-factor versus total risk management for the prevention of cardiovascular events in Seychelles.

OBJECTIVE: To assess the prevalence of cardiovascular (CV) risk factors in Seychelles, a middle-income African country, and compare the cost-effectiveness of single-risk-factor management (treating individuals with arterial blood pressure >/= 140/90 mmHg and/or total serum cholesterol >/= 6.2 mmol/l) with that of management based on total CV risk (treating individuals with a total CV risk >/= 10% or >/= 20%).METHODS: CV risk factor prevalence and a CV risk prediction chart for Africa were used to estimate the 10-year risk of suffering a fatal or non-fatal CV event among individuals aged 40-64 years. These figures were used to compare single-risk-factor management with total risk management in terms of the number of people requiring treatment to avert one CV event and the number of events potentially averted over 10 years. Treatment for patients with high total CV risk (>/= 20%) was assumed to consist of a fixed-dose combination of several drugs (polypill). Cost analyses were limited to medication.FINDINGS: A total CV risk of >/= 10% and >/= 20% was found among 10.8% and 5.1% of individuals, respectively. With single-risk-factor management, 60% of adults would need to be treated and 157 cardiovascular events per 100 000 population would be averted per year, as opposed to 5% of adults and 92 events with total CV risk management. Management based on high total CV risk optimizes the balance between the number requiring treatment and the number of CV events averted.CONCLUSION: Total CV risk management is much more cost-effective than single-risk-factor management. These findings are relevant for all countries, but especially for those economically and demographically similar to Seychelles.

Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

aIr-1, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHC class II gene expression.

RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.

Interleukin-1 receptor antagonist gene (IL-1RN) polymorphism is a predictive factor of clinical pregnancy after IVF.

BACKGROUND: Only 25% of IVF transfer cycles lead to a clinical pregnancy, calling for continued technical progress but also more in depth analysis of patients' individual characteristics. The interleukin-1 (IL-1) system and matrix metalloproteinases (MMPs) are strongly implicated in embryo implantation. The genes coding for IL-1Ra (gene symbol IL-1RN), IL-1beta, MMP2 and MMP9 bear functional polymorphisms. We analysed the maternal genetic profile at these polymorphic sites in IVF patients, to determine possible correlations with IVF outcome. METHODS: One hundred and sixty women undergoing an IVF cycle were enrolled and a buccal smear was obtained. The presence of IL-1RN variable number of tandem repeats and IL-1B + 3953, MMP2-1306 and MMP9-1562 single nucleotide substitutions were determined. Patients were divided into pregnancy failures (119), biochemical pregnancies (8) and clinical pregnancies (33). RESULTS: There was a 40% decrease in IL-1RN*2 allele frequency (P = 0.024) and a 45% decrease in IL-1RN*2 carrier status in the clinical pregnancy group as compared to the pregnancy failure group (P = 0.017). This decrease was still statistically significant after a multivariate logistic regression analysis. The likelihood of a clinical pregnancy was decreased accordingly in IL-1RN*2 carriers: odds ratio = 0.349, 95% confidence interval = 0.2-0.8, P = 0.017. The IL-1B, MMP2 and MMP9 polymorphisms showed no correlation with IVF outcome. CONCLUSIONS: IL-1RN*2 allele carriage is associated with a poor prognosis of achieving a pregnancy after IVF.

Beneficial effect of insulin-like growth factor-1 on hypoxemic renal dysfunction in the newborn rabbit.

Acute normocapnic hypoxemia can cause functional renal insufficiency by increasing renal vascular resistance (RVR), leading to renal hypoperfusion and decreased glomerular filtration rate (GFR). Insulin-like growth factor 1 (IGF-1) activity is low in fetuses and newborns and further decreases during hypoxia. IGF-1 administration to humans and adult animals induces pre- and postglomerular vasodilation, thereby increasing GFR and renal blood flow (RBF). A potential protective effect of IGF-1 on renal function was evaluated in newborn rabbits with hypoxemia-induced renal insufficiency. Renal function and hemodynamic parameters were assessed in 17 anesthetized and mechanically ventilated newborn rabbits. After hypoxemia stabilization, saline solution (time control) or IGF-1 (1 mg/kg) was given as an intravenous (i.v.) bolus, and renal function was determined for six 30-min periods. Normocapnic hypoxemia significantly increased RVR (+16%), leading to decreased GFR (-14%), RBF (-19%) and diuresis (-12%), with an increased filtration fraction (FF). Saline solution resulted in a worsening of parameters affected by hypoxemia. Contrarily, although mean blood pressure decreased slightly but significantly, IGF-1 prevented a further increase in RVR, with subsequent improvement of GFR, RBF and diuresis. FF indicated relative postglomerular vasodilation. Although hypoxemia-induced acute renal failure was not completely prevented, IGF-1 elicited efferent vasodilation, thereby precluding a further decline in renal function.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

Defective tumor necrosis factor-alpha-dependent control of astrocyte glutamate release in a transgenic mouse model of Alzheimer disease.

The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.

Factor shares, the price markup, and the elasticity of substitution between capital and labor

[spa] La participación del trabajo en la renta nacional es constante bajo los supuestos de una función de producción Cobb-Douglas y competencia perfecta. En este artículo se relajan estos supuestos y se investiga si el comportamiento no constante de la participación del trabajo en la renta nacional se explica por (i) una elasticidad de sustitución entre capital y trabajo no unitaria y (ii) competencia no perfecta en el mercado de producto. Nos centramos en España y los U.S. y estimamos una función de producción con elasticidad de sustitución constante y competencia imperfecta en el mercado de producto. El grado de competencia imperfecta se mide a través del cálculo del price markup basado en laaproximación dual. Mostramos que la elasticidad de sustitución es mayor que uno en España y menor que uno en los US. También mostramos que el price markup aleja la elasticidad de sustitución de uno, lo aumenta en España, lo reduce en los U.S. Estos resultados se utilizan para explicar la senda decreciente de la participación del trabajo en la renta nacional, común a ambas economías, y sus contrastadas sendas de capital.

Isolated limb perfusion: distinct tourniquet and tumor necrosis factor effects on the early hemodynamic response.

HYPOTHESIS: Recent evidence indicates that tumor response rates after isolated limb perfusion (ILP) are improved when tumor necrosis factor (TNF) is added to the locoregional perfusion of high doses of chemotherapy. Other factors, related to the patient or the ILP procedure, may interfere with the specific role of TNF in the early hemodynamic response after ILP with TNF and high-dose chemotherapy. DESIGN: Case-control study. SETTING: Tertiary care university hospital. PATIENTS: Thirty-eight patients with a locoregionally advanced tumor of a limb treated by ILP with TNF and high-dose chemotherapy (TNF group) were compared with 31 similar patients treated by ILP with high-dose chemotherapy alone (non-TNF group). INTERVENTIONS: Swan-Ganz catheter hemodynamic recordings, patients' treatment data collection, and TNF and interleukin 6 plasma level measurements at regular intervals during the first 36 hours following ILP. MAIN OUTCOME MEASURES: Hemodynamic profile and total fluid and catecholamine administration. RESULTS: In the TNF group, significant changes were observed (P<.006): the mean arterial pressure and the systemic vascular resistance index decreased, and the temperature, heart rate, and cardiac index increased. These hemodynamic alterations started when the ILP tourniquet was released (ie, when or shortly after the systemic TNF levels were the highest). The minimal mean arterial pressure, the minimal systemic vascular resistance index, the maximal cardiac index, the intensive care unit stay, and the interleukin 6 maximal systemic levels were significantly (P<.001 for all) correlated to the log(10) of the systemic TNF level. In the non-TNF group, only a brief decrease in the blood pressure following tourniquet release and an increase in the temperature and in the heart rate were statistically significant (P<.006). Despite significantly more fluid and catecholamine administration in the TNF group, the mean arterial pressure and the systemic vascular resistance index were significantly (P<.001) lower than in the non-TNF group. CONCLUSIONS: Release of the tourniquet induces a blood pressure decrease that lasts less than 1 hour in the absence of TNF and that is distinct from the septic shock-like hemodynamic profile following TNF administration. The systemic TNF levels are correlated to this hemodynamic response, which can be observed even at low TNF levels.