Reconstitution of ventricular myosin with atrial light chains 1 improves its functional properties

Atrial light chain 1 (ALC-1) is expressed in embryonic and hypertrophied human ventricles but not in normal adult human ventricles. We investigated the effects of recombinant human atrial light chains (hALC-1) on the structure and enzymatic activity of synthetic filaments of ventricular myosin. The endogenous ventricular myosin light chain 1 (VLC-1) was partially replaced by recombinant hALC-1 yielding hALC-1 levels of 12%, 24% and 42%. This reconstitution of ventricular myosin with hALC-1 did not change the length of synthetic myosin filaments but led to more rounded myosin heads in comparison with those of control filaments. Actin-activated ATPase activity of myosin, a parameter of functional activity of molecular motor, amounted to 79.5 nmol Pi/mg per min in control myosin filaments. Reconstitution with hALC-1 caused a profound increase of the actin-activated myosin ATPase activity in a dose dependent manner, for example, synthetic myosin filaments formed with 12%, 24% and 42% hALC-1 reconstituted myosin revealed the actin-activated ATPase activity increased by 18%, 26% and 36%, respectively, as compared to control. These results strongly suggest that in vivo expression of ALC-1 enhances ventricular myosin function, thereby contributing to cardiac compensation.     

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.     

A new C-nucleoside analogue of tiazofurin: synthesis and biological evaluation of 2-beta-D-ribofuranosylimidazole-4-carboxamide (imidazofurin)

2-Beta-D-ribofuranosylimidazole-4-carboxamide, an imidazole analogue of the antitumor agent tiazofurin, was synthesized and evaluated for the growth inhibitory activity of human myelogenous leukemia K562 cells.     

Cytosolic RNase inhibitor only affects RNases with intrinsic cytotoxicity

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.     

Bone growth, modeling and remodeling in a supernumerary metatarsal bone associated with segmental gigantism in cutis marmorata telangiectatica congenita

Skeletal structure and processes of bone growth, modeling and remodeling were studied in a supernumerary metatarsal surgically removed from a 3-year-old boy affected by Cutis Marmorata Telangiectatica Congenita (CMTC), associated with hypertrophy of the right upper and lower limbs and postaxial hexadactylism of the homolateral hand and foot. No other anomalies were observed. The excess of periosteal growth, due to congenital anomaly, induced an abnormal development of both modeling and remodeling processes. In bone modeling, osteoblast activity on the periosteal surface was not paralleled by osteoclast resorption along the wall of the medullary canal, and this enormously increased the cortical thickness. In bone remodeling, osteoclastic resorption cavities were not refilled by secondary Haversian systems, thus inducing a severe bone loss. While the alteration of bone growth and modeling can be ascribed to the congenital disease, the unbalanced bone remodeling appears mainly to depend on mechanical disuse of the supernumerary metatarsal.     

State-specific monoclonal antibodies identify an intermediate state in epsilon protein kinase C activation

Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.     

High frequency of TNF alleles -238A and -376A in individuals from northern Sardinia

The G to A single nucleotide polymorphisms (SNPs), at position -376, -308 and -238 in the promoter of the tumor necrosis factor alpha (TNF) gene, have been independently correlated with numerous diseases. Alleles TNF(-376A) and TNF(-238A) are normally found throughout the world with very low frequencies. We investigated the frequency of these SNPs in Sicilian subjects hospitalized after traumatic brain injury and in three groups of subjects from northern Sardinia: healthy subjects and individuals with multiple sclerosis or ischemic stroke. While no significant difference was found between healthy and disease subjects, the frequency of TNF(-376A) and TNF(-238A) was elevated up to 10 times in Sardinia compared to Sicily and other populations throughout the world. These elevated frequencies may be the result of genetic drift or of selective pressure on TNF itself or on neighboring genes, including the HLA. Malaria, endemic to Sardinia until the end of the 1940s, and the bubonic plague, are among the possible causes of selection. These findings indicate that Sardinia is an ideal location to further elucidate the correlation between TNF or HLA polymorphisms and diseases, including multiple sclerosis and type-I diabetes, present with an unusually high frequency and co-morbidity in Sardinia.     

Syntheses and biological evaluation of 5-(piperidin-1-yl)-3-phenyl-pentylsulfones as CCR5 antagonists

Cellular proliferation of HIV-1 requires the cooperative assistance of both the CCR5 and CD4 receptors. Our medicinal chemistry efforts in this area have resulted in the identification of N-alkyl piperidine sulfones as CCR5 antagonists. These compounds display potent binding and show antiviral properties in HIV-1 spread cell-based assays.     

PKC-{epsilon}-dependent survival signals in diabetic hearts

Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The epsilon-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-epsilon will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-epsilon translocation activator [psiepsilon-receptors for activated C kinase (psiepsilon-RACK)]. The results demonstrate a striking PKC-epsilon cardioprotective phenotype in diabetic psiepsilon-RACK (epsilon-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic epsilon-agonist mice exhibited selective trafficking of PKC-epsilon to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-epsilon in hearts of diabetic epsilon-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.