Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of Differentiation on the Leucine Enkephalin-Degrading Soluble Enzymes Released by the K562(S) Cell Line

Leu-enkephalin hydrolysis kinetics were measured in the presence of soluble supernatants obtained from cultures of the K562(S) leukaemic cell line. Under these conditions, the substrate is degraded with formation of two distinct patterns of the hydrolysis by-products: in one pattern, similar amounts of Tyr and Tyr-Gly are formed; in the other, only Tyr-Gly can be measured. Kinetic data suggest that soluble proteolyses are released by these cells, and that either dipeptidylaminopeptidases alone, or both aminopeptidases and dipeptidylaminopeptidases are involved in substrate hydrolysis. This alternation of hydrolysis patterns appears consistent with existing data on the heterogeneity of K562 cells. In contrast with these results, chromatographic separation of the soluble enzymes indicates the release of all three classes of proteolyses known to hydrolyze enkephalins: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In cells induced to differentiate by treatment with butyric acid, substrate hydrolysis is increased, and the pattern of the enzymes released is modified. In these cells, variations in both total proteolytic activity, and ratio between the three enzyme classes mentioned above are only minor, while the ratio between the different enzyme species within each class is greatly modified. Data obtained suggest that the expression of soluble enzymes is modified by differentiation. These data may also be interpreted as stressing the role of competition in controlling substrate hydrolysis by the multiple enzymes co-released by K562(S) cells.     

The role of osmiophilic bodies and Pfg377 expression in female gametocyte emergence and mosquito infectivity in the human malaria parasite Plasmodium falciparum

Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.     

Mood disorder in a patient with Smith-Magenis syndrome: a case report

Smith-Magenis syndrome (SMS) is a microdeletion syndrome characterized by physical and neurobehavioural features. This report describes the case of a 27 year old female affected by SMS associated with a diagnosis, according to DSMIV criteria, of Mood Disorder N.O.S. and Intermittent Explosive Disorder. To our knowledge, the association of SMS with mood shifts has never been reported. Considering the genetic alterations that characterizes the SMS, further investigations on the region of the chromosome 17p11.2 could help produce more information on the role of melatonin in the genesis of mood disorder.     

Paternal contribution to small for gestational age babies: a multicenter prospective study

Our aims were to investigate whether men who fathered small for gestational age (SGA) infants themselves had lower birthweight, were more likely to be obese, have central adiposity and elevated blood pressure in adult life compared with men who fathered non-SGA infants. A total of 2,002 couples participating in the Screening for Pregnancy Endpoints (SCOPE) study were enrolled in early pregnancy and pregnancy outcome data collected prospectively. SGA was defined as birthweight <10th customized centile, obesity as BMI ≥30 kg/m(2), central adiposity as waist circumference >102 cm. Logistic regression was used to compare rates of obesity, and central adiposity between men who fathered SGA infants compared with those with non-SGA infants and the final model was adjusted for maternal and paternal confounders. The men who fathered an SGA infant (209 (10.4%)) themselves had lower mean birthweight (3,291 (530) g vs. 3,472 (584) g, P < 0.0001), were more likely to be obese (50 (24.8%) vs. 321 (18.3%), adjusted odds ratio (OR) 1.50, 95% confidence interval 1.05-2.16, adjusted for maternal and paternal factors) and to have central adiposity (52 (25.1%) vs. 341 (19.2%), adjusted OR 1.53, 95% confidence interval 1.06-2.20) compared with men who fathered a non-SGA infant. Elevated paternal blood pressure was not associated with SGA. In conclusion, we report a novel relationship between paternal obesity/central adiposity and birth of an SGA infant, which appears to be independent of maternal factors associated with fetal growth restriction.     

Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.     

Co-culture of human fibroblasts,smooth muscle and endothelial cells promotes osteopontin induction in hypoxia

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.     

The Diadenosine Homodinucleotide P18 Improves In Vitro Myelination in Experimental Charcot‐Marie‐Tooth Type 1A

Charcot‐Marie‐Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca²⁺ concentration ([Ca²⁺]_i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca²⁺ levels through down‐regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5′,5′′′‐P1, P2‐diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11‐overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]_i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild‐type (wt) cells. P18 increased [cAMP]_i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non‐phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]_i‐increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype. J. Cell. Biochem. 115: 161–167, 2014. © 2013 Wiley Periodicals, Inc.     

Silicone-stimulated anthraquinone production and release by Morinda citrifolia in a two-liquid-phase system

Summary A 2.5-fold increase in the release of intracellular anthraquinones was obtained by adding 1 ml L^-1 silicone A to suspension cultures of Morinda citrifolia. Cell growth and secondary metabolite production were not affected even at high silicone A concentrations. Performance of the silicone treatment in a two-liquid-phase system (5 ml n-hexadecane/50 ml medium) resulted in a 150% increase of the overall secondary metabolite productivity.     

Effect of propylthiouracil-induced hypothyroidism on cerebral cortex of young and aged rats: Lipid composition of synaptosomes,muscarinic receptor sites,and acetylcholinesterase activity

The effect of hypothyroidism on the lipid composition of synaptosomes, density and affinity of muscarinic receptor sites, and acetylcholinesterase activity in the cerebral cortex of young and aged rats was investigated. The animals were made hypothyroid by adding 0.05% propyl-2-thiouracil to their drinking water for four weeks. This pathological state induced an increase in the relative percentage of sphingomyelin in young rats. In aged rats hypothyroidism induced a decrease of sphingomyelin and glycerophosphocholine and an increase of cholesterol. The effect of hypothyroid state on cerebral cortex resulted in an increase of acethylcholinesterase activity both in young and aged rats and was also reflected in an increase of density of M1-AChRs but only in the former.