Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence

We show that knockdown of 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) inhibits growth of lung cancer cells by senescence induction. This inhibition is not due to a defect in the oxidative PPP per se. NADPH and ribose phosphate production are normal in 6PGD knockdown cells and shutdown of PPP by knockdown of glucose-6-phosphate dehydrogenase (G6PD) has little effect on cell growth. Moreover, 6PGD knockdown cells can proliferate when the PPP is bypassed by using fructose instead of glucose in medium. Significantly, G6PD knockdown rescues proliferation of cells lacking 6PGD, suggesting an accumulation of growth inhibitory glucose metabolics in cells lacking 6PGD. Therefore, 6PGD inhibition may provide a novel strategy to treat glycolyic tumors such as lung cancer.     

Neuroendocrine-immune (NEI) circuitry from neuron-glial interactions to function: Focus on gender and HPA-HPG interactions on early programming of the NEI system

Bidirectional communication between the neuroendocrine and immune systems during ontogeny plays a pivotal role in programming the development of neuroendocrine and immune responses in adult life. Signals generated by the hypothalamic-pituitary-gonadal axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids), and by the hypothalamic-pituitary-adrenocortical axis (glucocorticoids (GC)), are major players coordinating the development of immune system function. Conversely, products generated by immune system activation exert a powerful and long-lasting regulation on neuroendocrine axes activity. The neuroendocrine-immune system is very sensitive to preperinatal experiences, including hormonal manipulations and immune challenges, which may influence the future predisposition to several disease entities. We review our work on the ongoing mutual regulation of neuroendocrine and immune cell activities, both at a cellular and molecular level. In the central nervous system, one chief compartment is represented by the astroglial cell and its mediators. Hence, neuron-glial signalling cascades dictate major changes in response to hormonal manipulations and pro-inflammatory triggers. The interplay between LHRH, sex steroids, GC and pro-inflammatory mediators in some physiological and pathological states, together with the potential clinical implications of these findings, are summarized. The overall study highlights the plasticity of this intersystem cross-talk for pharmacological targeting with drugs acting at the neuroendocrine-immune interface.     

Regulation of free radical outflow from an isolated muscle bed in exercising humans

Incremental knee extensor (KE) exercise performed at 25, 70, and 100% of single-leg maximal work rate (WR(MAX)) was combined with ex vivo electron paramagnetic resonance (EPR) spectroscopic detection of alpha-phenyl-tert-butylnitrone (PBN) adducts, lipid hydroperoxides (LH), and associated parameters in five males. Blood samples were taken from the femoral arterial and venous circulation that, when combined with measured changes in femoral venous blood flow, permitted a direct examination of oxidant exchange across a functionally isolated contracting muscle bed. KE exercise progressively increased the net outflow of LH and PBN adducts (100% > 70% > 25% WR(MAX), P < 0.05) consistent with the generation of secondary, lipid-derived oxygen (O(2))-centered alkoxyl and carbon-centered alkyl radicals. Radical outflow appeared to be more intimately associated with predicted decreases in intracellular Po(2) (iPo(2)) as opposed to measured increases in leg O(2) uptake, with greater outflow recorded between 25 and 70% WR(MAX) (P < 0.05 vs. 70-100% WR(MAX)). This bias was confirmed when radical venoarterial concentration differences were expressed relative to changes in the convective components of O(2) extraction and flow (25-70% WR(MAX) P < 0.05 vs. 70-100% WR(MAX), P > 0.05). Exercise also resulted in a net outflow of other potentially related redox-reactive parameters, including hydrogen ions, norepinephrine, myoglobin, lactate dehydrogenase, and uric acid, whereas exchange of lipid/lipoproteins, ascorbic acid, and selected lipid-soluble anti-oxidants was unremarkable. These findings provide direct evidence for an exercise intensity-dependent increase in free radical outflow across an active muscle bed that was associated with an increase in sarcolemmal membrane permeability. In addition to increased mitochondrial electron flux subsequent to an increase in O(2) extraction and flow, exercise-induced free radical generation may also be regulated by changes in iPo(2), hydrogen ion generation, norepinephrine autoxidation, peroxidation of damaged tissue, and xanthine oxidase activation.     

Expression of the apoptotic calcium channel P2X<Subscript>7</Subscript> in the glandular epithelium

In the current study, expression of the apoptotic calcium channel receptor P2X₇ and prostate-specific antigen (PSA) levels were studied in biopsy cores from 174 patients as well as 20 radical prostatectomy cases. In clinical biopsies, we have previously demonstrated that P2X₁ and P2X₂ calcium channel receptors are absent from normal prostate epithelium that does not progress to prostate cancer within 5 years. In cases that did progress to prostate cancer however, P2X₁ and P2X₂ labeling was observed in a stage-specific manner first in the nucleus, then the cytoplasm and finally on the apical epithelium, as prostate cancer developed. These markers were present up to 5years before cancer was detectable by the usual morphological criteria (Gleason grading) as determined by H&E staining. In the current study, the apoptotic calcium channel receptor P2X₇ yielded similar results to that of P2X₁ and P2X₂. Using radical prostatectomy tissue sections as well as biopsies, these changes in calcium channel metabolism were noted throughout the prostate, indicating a field effect. This finding suggests that the presence of a prostate tumor could be detected without the need for direct sampling of tumor tissue, leading to detection of false negative cases missed by H&E stain. The reliability of PSA levels as a prognostic indicator has been questioned in recent years. In the current study, PSA levels were correlated with the P2X₇ labeling results. All patients who exhibited no P2X₇ labeling had a prostatic serum antigen (PSA) level of <2. Patients who exhibited stage-specific P2X₇ expression, and who later developed obvious prostate cancer as diagnosed by H&E stain, all had a PSA > 2. This finding suggests that increasing PSA may be an accurate indicator of cancer development.     

Effect of transient neonatal hypothyroidism on the free aspartic acid, glutamic acid, and GABA content of different central auditory regions in the rat

The aspartic acid, glutamic acid, and gamma-aminobutyric acid (GABA) contents were determined in four central auditory system regions in rats with transient neonatal hypothyroidism compared with control ones: the ventral and dorsal parts of the cochlear nucleus, the central nucleus of the inferior colliculus, the auditory cortex, and in an extra-auditory structure, the substantia nigra pars reticulata. The animals were sacrificed at 50 days of age, brain tissue samples were taken out by microdissection, and the free amino acids were extracted. The amino acid content was assessed by double-isotope labelling following two-dimensional thin-layer chromatography separation. GABA content was significantly decreased in both cochlear nucleus regions and glutamic acid was elevated in the inferior colliculus. Neonatal hypothyroidism had no significant effect on the aspartic acid levels in the regions studied. The results suggest an effect of neonatal hypothyroidism on regional contents of free amino acids known as candidate neurotransmitters in the auditory system.     

The physical attractiveness of facially deformed patients before and after craniofacial surgery

The present experiment investigated whether the physical attractiveness of craniofacially deformed children and adolescents could be improved by surgical procedures. Twenty patients between the ages of 5 months and 17 years were randomly selected from patient files. Patient diagnoses included facial clefts, hypertelorism, Treacher Collins syndrome, and craniofacial dysostosis (Crouzon's and Apert's syndromes). Rigorously standardized photographs of patients taken before and after surgery were shown to 40 "naive" raters ranging in age from 17 to 52 years. Raters analyzed the photographs with regard to global physical attractiveness. These ratings indicated that the patients' physical attractiveness was reliably (62 percent) improved following surgery. The results are discussed in light of recent evidence that untreated craniofacial patients may be at risk for psychosocial disorders and in terms of the growing evidence of the importance of physical appearance for the development of cognitive and social-emotional competence. In addition, a standardized assessment system is described that can be used to facilitate the compilation of actuarial data predicting surgical outcomes. Finally, the importance of empirically evaluating the effectiveness of surgical procedures and practitioners on a continuing basis is emphasized.     

The oxidative generation of sulfonic acid groups in neuromelanin and lipofuscin in the human brain

Sulfonic acid groups were oxidatively generated in the soluble lipid-free lipofuscin component of neuromelanin of human substantia nigra and in lipofuscin of human inferior olive. Exposure of these oxidized, intraneuronal pigments to low pH Alcian blue or aldehyde fuchsin demonstrated an intensity of staining that related to the type of oxidant and the conditions of its use. Utilization of the following oxidants generated increasingly strong staining reactions as signified by the following sequence; periodic acid under mild conditions, bromine in carbon tetrachloride, hydrogen peroxide, periodic acid under drastic conditions, potassium permanganate followed by oxalic acid, hydrogen peroxide followed by bromine in carbon tetrachloride, potassium permanganate followed by metabisulfite or bisulfite, and performic acid. Neither Alcian blue nor aldehyde fuchsin revealed oxidatively generated aldehyde as judged by 1) their failure or near failure to stain inferior olive lipofuscin following mildly applied periodic acid, and 2) the increase in staining intensity, from moderate to strong, displayed by the soluble lipid-free lipofuscin component of neuromelanin and by inferior olive lipofuscin when potassium permanganate was followed by a rinse with metabisulfite or bisulfite in place of one with oxalic acid.     

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Immunohistochemical characterization of monolayer cell cultures of embryonic chicken pancreas and measurement of somatostatin release.

Monolayer cell cultures of embryonic chicken pancreas contain functionally active insulin, glucagon and somatostatin-containing cells as evidenced by immunohistochemical and radioimmunoassay techniques. Hormone release is in relation to the number of each cell type present and responds to known specific secretory stimuli. The relatively high numbers of D-cells and amounts of immunoreactive somatostatin released by this preparation makes this system a suitable model for studies of somatostatin function and secretion.     

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.