Cell cycling and differentiation do not require the retinoblastoma protein during early Xenopus development

The retinoblastoma protein (pRb) is a central regulator of the cell cycle, controlling passage through G1 phase. Moreover, pRb has also been shown to play a direct role in the differentiation of multiple tissues, including nerve and muscle. Rb null mice display embryonic lethality, although recent data have indicated that at least some of these defects are due to placental insufficiency. To investigate this further, we have examined the role of pRb in early development of the frog Xenopus laevis, which develops without the need for a placenta. Surprisingly, we see that loss of pXRb has no effect on either cell cycling or differentiation of neural or muscle tissue, while overexpression of pXRb similarly has no effects. We demonstrate that, in fact, pXRb is maintained in a hyperphosphorylated and therefore inactive state early in development. Therefore, Rb protein is not required for cell cycle control or differentiation in early embryos, indicating unusual control of these G1/G0 events at this developmental stage.     

Neuropeptide Y and peptide YY neuronal and endocrine systems

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.     

Pulsatile and nocturnal growth hormone secretions in men do not require periodic declines of somatostatin

Using a continuous subcutaneous octreotide infusion to create constant supraphysiological somatostatinergic tone, we have previously shown that growth hormone (GH) pulse generation in women is independent of endogenous somatostatin (SRIH) declines. Generalization of these results to men is problematic, because GH regulation is sexually dimorphic. We have therefore studied nine healthy young men (age 26 +/- 6 yr, body mass index 23.3 +/- 1.2 kg/m2) during normal saline and octreotide infusion (8.4 microg/h) that provided stable plasma octreotide levels (764.5 +/- 11.6 pg/ml). GH was measured in blood samples obtained every 10 min for 24 h. Octreotide suppressed 24-h mean GH by 52 +/- 13% (P = 0.016), GH pulse amplitude by 47 +/- 12% (P = 0.012), and trough GH by 39 +/- 12% (P = 0.030), whereas GH pulse frequency and the diurnal rhythm of GH secretion remained essentially unchanged. The response of GH to GH-releasing hormone (GHRH) was suppressed by 38 +/- 15% (P = 0.012), but the GH response to GH-releasing peptide-2 was unaffected. We conclude that, in men as in women, declines in hypothalamic SRIH secretion are not required for pulse generation and are not the cause of the nocturnal augmentation of GH secretion. We propose that GH pulses are driven primarily by GHRH, whereas ghrelin might be responsible for the diurnal rhythm of GH.     

Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut

Endocrine cells containing peptide YY (PYY) were numerous in the rectum, colon and ileum and few in the duodenum and jejunum of rat, pig and man. No immunoreactive cells could be detected in the pancreas and stomach. Coexistence of PYY and glicentin was revealed by sequential staining of the same section and by staining consecutive semi-thin sections. Since the PYY sequence is not contained in the glucagon/glicentin precursor molecule the results suggest that the PYY cell in the gut expresses two different genes coding for regulatory peptides of two different families.     

Luteal enzymes of the luteinizing hormone and beta-adrenergic signal transduction pathways in hypophysectomized rabbits do not require pituitary hormone support.

Experiments were conducted to determine whether continuous pituitary hormone support is required for expression of the LH and beta-adrenergic cAMP signal transduction pathways in rabbit CL during pseudopregnancy. Parameters of the LH and catecholamine cAMP signal transduction pathways in CL of estrogen-treated hypophysectomized rabbits were compared to those of pituitary-intact rabbits. Results showed that each of the parameters of the LH and beta-adrenergic cAMP signal transduction pathways was retained in CL taken from estrogen-treated pseudopregnant rabbits that had been hypophysectomized for as long as 13 days at levels not significantly different from those of estrogen-treated pituitary-intact rabbits. These included luteal basal, and LH-, epinephrine-, and fluoride-stimulated adenylyl cyclase activities; total luteal cAMP levels; the number and affinity of cAMP-dependent protein kinase regulatory subunit cAMP binding sites; binding activity of the type I and type II regulatory subunits; and the amount of catalytic subunit protein of cAMP-dependent protein kinase. We conclude that expression of the proteins of the cAMP signal pathway for LH and beta-adrenergic hormones in CL of estrogen-treated rabbits does not require pituitary hormone support.     

Distribution of enteric neural peptide YY in the dog gastrointestinal tract

Various regions of the dog gastrointestinal tract were investigated for the distribution of peptide YY (PYY) neurons using immunocytochemistry and radioimmunoassay. PYY neurons that encircled non-PYY-immunoreactive neurons were mainly observed in the myenteric plexus from the stomach to the colon. There was more PYY-like immunoreactivity in the muscle layer of the stomach and ileum than in the other intestines. The results of high performance liquid chromatography revealed that neural PYY-immunoreactive substance is identical to authentic PYY. PYY was not localized in the cholinergic neurons. These results indicate that PYY, as a neuropeptide, is involved in the regulation of gastrointestinal function.     

Immunohistochemical study on the ontogenetic development of the regional distribution of peptide YY, pancreatic polypeptide, and glucagon-like peptide 1 endocrine cells in bovine gastrointestinal tract

The regional distribution and relative frequency of peptide YY (PYY)-, pancreatic polypeptide (PP)-, and glucagon-like peptide 1 (GLP-1)-immunoreactive (IR) cells were determined immunohistochemically in the gastrointestinal tract at seven ontogenetic stages in pre- and postnatal cattle. Different frequencies of PYY-, PP-, and GLP-1-IR cells were found in the intestines at all stages; they were not found in the esophagus and stomach. The frequencies varied depending on the intestinal segment and the developmental stage. The frequencies of PYY- and PP-IR cells were lower in the small intestine and increased from ileum to rectum, whereas GLP-1-IR cells were more numerous in duodenum and jejunum, decreased in ileum and cecum, and increased again in colon and rectum. The frequencies also varied according to pre- and postnatal stages. All three cell types were most numerous in fetus, and decreased in calf and adult groups, indicating that the frequencies of these three types of endocrine cells decrease with postnatal development. The results suggest that these changes vary depending on feeding habits and adaptation of growth, secretion, and motility of intestine at different ontogenetic stages of cattle.     

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.     

Phosphatidylethanolamine biosynthesis in rat mammary carcinoma cells that require and do not require ethanolamine for proliferation

Epithelial cells and some of their transformed derivatives require ethanolamine to grow normally in defined culture medium. When these cells are cultured without ethanolamine, the amount of cellular phosphatidylethanolamine is considerably reduced. Using a set of rat mammary carcinoma cell lines whose growth is responsive (64-24 cells) and not responsive (22-1 cells) to ethanolamine, the biochemical mechanism of ethanolamine responsiveness was investigated. The biosynthesis and metabolism of phospholipid, particularly of those involving phosphatidylethanolamine, were thus compared between the two types of cells. The incorporation of [3H]serine into phosphatidylserine and phosphatidylethanolamine in 64-24 cells was 60 and 37%, respectively, of those in 22-1 cells. However, the activity of phosphatidylserine decarboxylase was virtually the same in these cell lines. When these cells were cultured in the presence of [32P]phosphatidylcholine and [32P]phosphatidylethanolamine, the rate of accumulation of 32P-labeled phosphatidylserine from the radioactive phosphatidylethanolamine was considerably reduced in 64-24 cells compared to that in 22-1 cells, although the rate of synthesis of phosphatidylserine and phosphatidylethanolamine from the radioactive phosphatidylcholine was similar between the two cell lines. The rate of labeling phosphatidylcholine from the radioactive phosphatidylethanolamine was also reduced in 64-24 cells, although the difference was not as great as that of phosphatidylserine. Incorporation of 32P into phosphatidylethanolamine was correlated with the concentration of ethanolamine in the culture medium in 64-24 cells, whereas in 22-1 cells the incorporation was not influenced by ethanolamine. Enzyme activities of the CDP-ethanolamine pathway were not significantly different between the two cell lines. The rate of degradation of phosphatidylethanolamine was also similar in these cell lines. These results show that ethanolamine responsiveness of 64-24 cells, and probably other epithelial cells, is due to a limited ability to synthesize phosphatidylserine resulting from a limited base-exchange activity utilizing phosphatidylethanolamine.     

Physiological evidence for the involvement of peptide YY in the regulation of energy homeostasis in humans

Objective: To explore the potential role of the endogenous peptide YY (PYY) in the long‐term regulation of body weight and energy homeostasis. Research Methods and Procedures: Fasting and postprandial plasma PYY concentrations were measured after an overnight fast and 30 to 180 minutes after a standardized meal in 29 (21 men/8 women) non‐diabetic subjects, 16 of whom had a follow‐up visit 10.8 ± 1.4 months later. Ratings of hunger and satiety were collected using visual analog scales. Resting metabolic rate (RMR) (15‐hour RMR) and respiratory quotient (RQ) were assessed using a respiratory chamber. Results: Fasting PYY concentrations were negatively correlated with various markers of adiposity and negatively associated with 15‐hour RMR (r = ?0.46, p = 0.01). Postprandial changes in PYY (area under the curve) were positively associated with postprandial changes in ratings of satiety (r = 0.47, p = 0.01). The maximal PYY concentrations achieved after the meal (peak PYY) were negatively associated with 24‐hour RQ (r = ?0.41, p = 0.03). Prospectively, the peak PYY concentrations were negatively associated with changes in body weight (r = ?0.58, p = 0.01). Discussion: Our data indicate that the endogenous PYY may be involved in the long‐term regulation of body weight. It seems that this long‐term effect was not exclusively driven by the modulation of food intake but also by the control of energy expenditure and lipid metabolism.     

Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey

The objective of this study was to characterize the distribution and concentration of peptide YY (PYY) in the gastrointestinal tract of the rat, dog, and monkey. In the rat, the greatest concentration of PYY was detected in the ileum and colon. The concentrations of PYY in the ileum and colon were 72 +/- 49 and 768 +/- 180 ng/g tissue, respectively. In the dog, PYY was found primarily in extracts of the mucosal layer of the ileum and colon, with smaller amounts in the distal jejunum. The concentration of PYY in the mucosal layers of the canine distal jejunum was 113 +/- 25 ng/g tissue, proximal jejunum 302 +/- 56, mid jejunum 507 +/- 151, distal ileum 691 +/- 184, and colon 1706 +/- 774 ng/g tissue. In the monkey gastrointestinal tract, PYY was detected predominantly in mucosal extracts of the jejunum, ileum, and colon. The concentration of PYY in the mucosal layer extract of the jejunum was 92 +/- 23, ileum 615 +/- 127, and colon 1013 +/- 243 ng/g tissue.     

Repeated administration of the anorectic factor prolactin-releasing peptide leads to tolerance to its effects on energy homeostasis

Central administration of a single dose of prolactin-releasing peptide (PrRP) causes a reduction in both fast-induced and nocturnal food intake and body weight gain. The aim of this study was to examine the effect of repeated administration of PrRP on energy homeostasis, including a measure of the expression of the mitochondrial uncoupling protein-1 (UCP-1) in brown adipose tissue. Conscious, free-feeding animals received central injections of PrRP (4 nmol icv) or vehicle. A single injection at 1000 caused a sustained hyperthermia over the 4-h test period and an increase in the expression of UCP-1 mRNA. Repeated, twice daily injection caused a reduction in body weight gain greater than that seen in pair-fed animals for the first 48-72 h. After 72 h, the animals became refractory to the actions of PrRP. The pair-fed group showed a reduction in UCP-1 mRNA expression at 48 h, which was reversed by PrRP treatment. This study indicates that PrRP exerts its effects on energy homeostasis in the short-medium term by reducing food intake and increasing energy expenditure.     

Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation

During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes - too few to surround a microtubule - were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.     

Closure of the uterine lumen and the plasma membrane transformation do not require blastocyst implantation

Ultrastructural changes in the plasma membrane of uterine epithelial cells in the pseudopregnant rat were examined to determine if these changes resemble those found during normal pregnancy and also to examine if the well-known membrane alterations of early pregnancy are intrinsic to uterine epithelial cells. Changes in the surface contours of uterine epithelial cells from the afternoon of day 6 to the morning of day 9 of pseudopregnancy were similar to those present after attachment in normal pregnancy although somewhat delayed. The presence of short, irregular microvilli was seen from as early as day 7 of pseudopregnancy, with regular microvilli returning to the epithelial surface by days 8-9 of pseudopregnancy but to a slightly lesser extent as compared to normal pregnancy. Furthermore, observations made on the afternoon of day 6 to the morning of day 7 of pseudopregnancy showed that the uterine lumen was closed down and that complete membrane flattening between opposing uterine epithelial cells was seen all along the uterus in the absence of a blastocyst. These observations establish that the "plasma membrane transformation" does not depend on blastocyst implantation.     

The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein Raidd

The receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD/CRADD) functions as a dual adaptor and is a constituent of different multi-protein complexes implicated in the regulation of inflammation and cell death. Within the PIDDosome complex, RAIDD connects the cell death-related protease, Caspase-2, with the p53-induced protein with a death domain 1 (PIDD1). As such, RAIDD has been implicated in DNA-damage-induced apoptosis as well as in tumorigenesis. As loss of Caspase-2 leads to an acceleration of tumor onset in the Eμ-Myc mouse lymphoma model, whereas loss of Pidd1 actually delays onset of this disease, we set out to interrogate the role of Raidd in cancer in more detail. Our data obtained analyzing Eμ-Myc/Raidd^−/− mice indicate that Raidd is unable to protect from c-Myc-driven lymphomagenesis. Similarly, we failed to observe a modulatory effect of Raidd deficiency on DNA-damage-driven cancer. The role of Caspase-2 as a tumor suppressor and that of Pidd1 as a tumor promoter can therefore be uncoupled from their ability to interact with the Raidd scaffold, pointing toward the existence of alternative signaling modules engaging these two proteins in this context.A number of mechanisms have evolved to trace and remove potentially dangerous cells. Deregulation of the induction of apoptosis upon oncogenic stress, for example, can facilitate the accumulation of cells prone to undergo malignant transformation. Cell death by apoptosis depends on the cascade-like activation of proteases of the Caspase family.¹ Among these, the evolutionarily most conserved protease, Caspase-2, turns out to be a potent tumor suppressor in mice^{2, 3, 4, 5, 6, 7} and correlative expression data support a conserved role in human cancer.^{8, 9, 10, 11, 12, 13}Early studies suggested that Caspase-2 interacts with other proteins for its activation (e.g., after genotoxic stress), but the protease seems also able to auto-activate cell death on its own when present in sufficiently high concentration.^{14, 15, 16, 17, 18} The most prominent Caspase-2-containing protein complex was dubbed the ‘PIDDosome'' and described to contain the p53-induced protein with a death domain (PIDD1) and receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD, also known as CRADD).¹⁹ Although the molecular details of the pro-apoptotic potential of Caspase-2 are still discussed and alternative roles in the DNA-damage response, cell cycle arrest or sensor of metabolic stress are mechanistically poorly understood, Caspase-2 clearly limits tumorigenesis in different settings. These include aberrant expression of c-Myc in B cells^{3, 4} or deletion of the DNA-damage response regulator, ataxia telangiectasia mutated kinase (ATM), both driving lymphomagenesis⁶ as well as overexpression of the Her2/ErbB2 oncogene in breast⁵ or that of mutated KRAS in the lung epithelium, driving carcinoma formation.⁷ One of these studies, addressing also the role of Pidd1 in c-Myc-driven lymphomagenesis, revealed an unexpected oncogenic role for Pidd1, thereby questioning the physiological relevance of the PIDDosome complex in Caspase-2-mediated cell death and tumor suppression.^{4, 20} However, the exact role of the scaffold protein Raidd within these processes remains unaddressed so far.Raidd, a bipartite adapter containing a death domain (DD) and a caspase-recruitment domain (CARD) was first described to bind to the DD-containing kinase RIPK1 and the C. elegans caspase CED-3,²¹ supporting a role in cell death initiation. Subsequently, the interaction of Caspase-2 and Raidd was biochemically proven²² and proposed to be required for Caspase-2 autoprocessing preceding its activation.¹⁹ More recent studies propose an anti-inflammatory role for Raidd through suppression of nuclear factor kappa-light-chain enhancer (NF-κB) activation and cytokine production upon T-cell receptor stimulation by negatively interfering with the Carma1/Malt1/Bcl-10 signaling complex.^{23, 24}First evidence for a potential role of RAIDD in human cancer was discovered in a biochemical screen using mantle cell lymphomas, which detected a downregulation of RAIDD by microarray analysis,¹⁰ whereas others reported on RAIDD-linked multidrug resistance in osteosarcoma cells.²⁵ Furthermore, tumor cell apoptosis induced by inhibitors of histone de-acetylases in treatment-resistant adult T-cell leukemia lines reportedly required Caspase-2 and Raidd.²⁶ It is also reported that the Caspase-2/Raidd axis is necessary after ER stress, for example, in the course of infection with the oncolytic maraba virus.²⁷Taken together, these studies support a role for RAIDD in drug-induced cancer cell death as well as in tumor suppression, most likely linked to its role as a direct activator of Caspase-2. Alternatively, RAIDD may negatively interfere with PIDD- or BCL10-regulated NF-κB signaling^{23, 24, 28} and thereby suppress pro-tumorigenic inflammation. To address the role of Raidd in tumorigenesis in more detail, we exploited different mouse models where we induced thymic lymphomas by γ-irradiation, fibrosarcomas by 3-methylcholanthrene (3-MC) injection or B-cell lymphomas by aberrant expression of the c-Myc proto-oncogene. Our results suggest that Raidd is not a suppressor of tumors in the mouse models tested.     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

Catabolite and transient repression in Escherichia coli do not require enzyme I of the phosphotransferase system.

Transient and catabolite repression with changes in intracellular concentrations of cyclic adenosine 3',5-monophosphate is produced by glycerol and by glucose-6-phosphate in a strain with a partial deletion of the structural gene for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.