An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and l-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of l-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP]². When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of l-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in l-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.     

Cell proliferation, apoptosis, NF-kappaB expression, enzyme, protein, and weight changes in livers of burned rats

Thermal injury has been shown to alter gut epithelium and heart myocyte homeostasis by inducing programmed cell death. The effect of thermal injury on hepatocyte apoptosis and proliferation, however, has not been established. The purpose of this study was to determine whether a large thermal injury increases liver cell apoptosis and proliferation and whether these changes were associated with alterations in hepatic nuclear factor kappaB (NF-kappaB) expression and changes in liver enzymes and amount of protein. Sprague-Dawley rats received a 40% total body surface area scald burn or sham burn. Rats were killed and livers were harvested at 1, 2, 5, and 7 days after burn. Liver cell apoptosis was determined by terminal deoxyuridine nick end labeling (TUNEL) assay and cell proliferation by immunohistochemistry for proliferating cell nuclear antigen. Hepatic NF-kappaB expression was determined by Western blot, and total hepatic protein content was determined by protein assay. Protein concentration decreased after burn compared with sham controls (P < 0.05). Liver cell apoptosis, proliferation, and NF-kappaB expression in hepatocytes increased in burned rats compared with controls (P < 0.05). It was concluded that thermal injury induces hepatic cell apoptosis and proliferation associated with an increase in hepatic NF-kappaB expression and a decrease in hepatic protein concentration.     

Solid-phase synthesis of the native sequence of horse heart cytochrome c-(66-104)-nonatriacontapeptide and of a C-terminal carboxyamide analog selectively modified at Met-80

The peptide corresponding to the (66-104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid-phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66-104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse greater than 65] (1-65)H heme-sequence.     

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy

Aims

Activation of Ca²⁺/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods and Results

In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion

These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.     

Antibody informatics for drug discovery

More and more antibody therapeutics are being approved every year, mainly due to their high efficacy and antigen selectivity. However, it is still difficult to identify the antigen, and thereby the function, of an antibody if no other information is available. There are obstacles inherent to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii) antibody numbering and IMGT. Here, we review “antibody informatics,” which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Mining cancer biology through bioinformatic analysis of proteomic data

ABSTRACT

Introduction: Discovery proteomics for cancer research generates complex datasets of diagnostic, prognostic, and therapeutic significance in human cancer. With the advent of high-resolution mass spectrometers, able to identify thousands of proteins in complex biological samples, only the application of bioinformatics can lead to the interpretation of data which can be relevant for cancer research.

Areas covered: Here, we give an overview of the current bioinformatic tools used in cancer proteomics. Moreover, we describe their applications in cancer proteomics studies of cell lines, serum, and tissues, highlighting recent results and critically evaluating their outcomes.

Expert opinion: The use of bioinformatic tools is a fundamental step in order to manage the large amount of proteins (from hundreds to thousands) that can be identified and quantified in a cancer biological samples by proteomics. To handle this challenge and obtain useful data for translational medicine, it is important the combined use of different bioinformatic tools. Moreover, a particular attention to the global experimental design, and the integration of multidisciplinary skills are essential for best setting of tool parameters and best interpretation of bioinformatics output.