Protein kinase C and phospholipase D: intimate interactions in intracellular signaling

Diacylglycerol (DAG) was discovered as a potent lipid second messenger with protein kinase C (PKC) as its major cellular target more than 25 years ago. There is increasing evidence of significant complexity within lipid signaling, and the classical DAG-PKC model no longer stands alone but is part of a larger bioactive lipid universe involving glycerolipids and sphingolipids. Multiple layers of regulation exist among PKC- and DAG-metabolizing enzymes such as phosphatidylcholine (PC)-specific phospholipase D, and cross-talk exists between the glycerolipid and sphingolipid pathways, with PKC at the center. Currently, there is intense interest in the question of whether DAG derived from PC can function as a lipid second messenger and regulate PKC analogous to DAG derived from phosphatidylinositol-4,5-bisphosphate (PIP₂). To address these issues and incorporate DAG-PKC and other signaling pathways into an expanded view of cell biology, it will be necessary to go beyond the classical approaches and concepts.Received 29 November 2004; received after revision 18 January 2005; accepted 4 March 2005This work is dedicated to the memory of Dr. Yasutomi Nishizuka, the discoverer of protein kinase C, who was both a gentleman and a scientist.     

Cellular responses to mild heat stress

Since its discovery in 1962 by Ritossa, the heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to heat stress. The most well characterized heat shock response is induction of the heat shock proteins that function as molecular chaperones and exert cell cycle regulatory and anti-apoptotic activities. While most investigators have focused their studies on the toxic effects of heat stress in organisms such as severe heat stress-induced cell cycle arrest and apoptosis, the cellular response to fever-ranged mild heat stress has been rather underestimated. However, the cellular response to mild heat stress is likely to be more important in a physiological sense than that to severe heat stress because the body temperature of homeothermic animals increases by only 1–2°C during febrile diseases. Here we provide information that mild heat stress does have some beneficial role in organisms via positively regulating cell proliferation and differentiation, and immune response in mammalian cells.Received 14 May 2004; received after revision 2 August 2004; accepted 16 August 2004     

Human peripheral blood monuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro

The efficiency of test vaccines needs to be evaluated by quantification of the triggered cellular immune response. Usually, for these assays, autologous target cells expressing the vaccine antigen are required. In the context of messenger RNA (mRNA)-based vaccinations, the target cells used for the read-out are mRNA-transfected monocyte-derived dendritic cells (Mo-DCs). Their production typically requires samples of 100 ml blood from the patients, and limits the number of assays that can be performed. We show here that fresh peripheral blood mononuclear cells (PBMCs) can be transfected with mRNA by electroporation. Such cells are as efficient as mRNA-transfected Mo-DCs for their ability to activate memory T cells in vitro. Thus, mRNA-transfected PBMCs are a convenient replacement of mRNA-transfected Mo-DCs for the in vitro monitoring of natural or vaccine-induced immune responses.Received 17 February 2005; received after revision 1 May 2005; accepted 7 Juni 2005     

Phosphodiesterase: overview of protein structures, potential therapeutic applications and recent progress in drug development

Phosphodiesterases (PDEs) are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Because of their great market potential and therapeutic importance, PDE inhibitors became recognized as important therapeutic agents in the treatment of various diseases. Currently, there are seven PDE inhibitors on the market, and the pharmacological and safety evaluations of many drug candidates are in progress. Three-dimensional (3D) structures of catalytic domains of PDE 1, -3, -4, -5 and -9 in the presence of their inhibitors are now available, and can be utilized for rational drug design. Recent advances in molecular pharmacology of PDE isoenzymes resulted in identification of new potential applications of PDE inhibitors in various therapeutic areas, including dementia, depression and schizophrenia. This review will describe the latest advances in PDE research on 3D structural studies, the potential of therapeutic applications and the development of drug candidates.Received 30 November 2004; received after revision 24 January 2005; accepted 5 February 2005     

Decision-making at the surface of the intact or barrier disrupted skin: potential applications for vaccination or therapy

The skin is a highly accessible organ and constitutes an active immunological site. Both these properties make this surface an attractive route for what promises to be a cost-effective, simple, practical and needle-free delivery of vaccines and immunomodulators. Less obvious is the fact that the state of the skin barrier can influence quantitative and qualitative aspects of antigen-specific immune responses. The everyday decision-making at the skin epithelium concerns the choice between the induction of an immune response and the establishment of a state of non-responsiveness (tolerance). This decision is influenced by various factors such as the dose, the route (intact vs barrier-disrupted skin), the cytokine microenvironment and the nature of the antigenic stimulus. By increasing our understanding of how immune responses are regulated in the epidermis we can envisage the development of immunisation protocols aimed at eliciting a protective immune response or inducing tolerance, with direct applications to preventive or therapeutic vaccination, respectively.Received 29 November 2004; received after revision 2 February 2005; accepted 22 February 2005     

Hsp70 chaperones: Cellular functions and molecular mechanism

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.Received 21 October 2004; received after revision 24 November 2004; accepted 6 December 2004     

Congenital muscular dystrophy: molecular and cellular aspects

The congenital muscular dystrophies are a clinically and genetically heterogeneous group of neuromuscular disorders. Each form has a characteristic phenotype, but there is overlap between some entities and their classification is based on a combination of clinical features and the primary or secondary protein defect. Recent studies have identified the genetic basis of a number of congenital muscular dystrophies (11 genes in total) and have recognised a novel pathological mechanism that highlights the importance of the correct posttranslational processing of proteins, in particular -dystroglycan. Diagnosis of these conditions has been aided by the availability of specific antibodies for each protein and a better understanding of the protein changes that accompany each condition. In this review we present the major molecular, clinical and diagnostic aspects of each group of congenital muscular dystrophy with an emphasis in the more recent developments.Received 11 December 2004; accepted 15 December 2004     

Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment

Recent reports describe successful treatment using copper chelation therapy in neurodegenerative animal models. However, the success claimed for chelation therapy in neurodegenerative diseases is still rather controversial. To acquire new information on copper metabolism/homeostasis, we utilized cuprizone, a very sensitive and selective copper-chelating agent with well-known neurotoxic properties, as a relevant chemical model in mice. Upon cuprizone treatment, mice developed a pronounced astrocytosis, with brain oedema and spongiosis characterised by vacuolisations of the neuropil predominantly in the white matter. In addition, cuprizone treatment severely altered copper and zinc homeostasis in the central nervous system (CNS) as well as in all other tissues examined, with increasing metal ion concentrations particularly in the CNS. Concomitant with this increase in the Cu and Zn concentration in the brain, metallothionein-I and -II were also highly immunoreactive in astrocyte, consistent with the astrocytosis and demyelination observed in our and other laboratories.Received 23 February 2005; received after revision 3 May 2005; accepted 13 May 2005     

Elucidation of the relationship between enzyme activity and internal motion using a lysozyme stabilized by cavity-filling mutations

We investigated the activity and the internal motions of a stabilized mutant hen lysozyme (HEL) in which the residues M12 and L56 were mutated to L and F, respectively (LF mutant HEL). The result of the activity measurements against glycol chitin at various temperatures suggested that the temperature dependence of the activity of LF mutant HEL shifted to the high-temperature side compared with that of wild-type HEL. The detailed internal motions of LF mutant HEL in the absence and presence of a substrate analogue, (NAG)₃, were examined by model-free analysis at 35°C. The results showed that the internal motions of LF mutant HEL in the presence of (NAG)₃ were drastically restricted compared with those in wild-type HEL. Our findings thus suggested that the mutation to the stabilized lysozyme restricted internal motions required for the enzymatic reaction.Received 8 February 2005; accepted 10 March 2005Y. Yoshida and T. Ohkuri contributed equally to this work.     

Genetic analysis of the kinome and phosphatome in cancer

Protein phosphorylation is a well-characterized biochemical process for reversible regulation of protein activity. Protein kinases and protein phosphatases are the key complementary players in this process, and through their coordinated activity cell homeostasis is tightly controlled. If these enzymes display aberrant activity, cells may undergo unrestrained growth, thus giving rise to complex diseases such as cancer. The technological platform gathered during the Human Genome Project recently allowed the systematic identifi cation of the genetic alterations present in the kinase (the kinome) and the phosphatase (the phosphatome) gene families. These studies suggest that most if not all human tumors carry genetic alterations in at least one phosphatase or kinase gene. Here we integrate the biochemical knowledge on the properties of these molecules with the information collected through their systematic genetic analysis in cancer. We also analyze why the molecular profi ling of the kinome and phosphatome in individual cancers is revolutionizing basic and clinical oncology.Received 13 May 2005; received after revision 30 May 2005; accepted 22 June 2005