Prereplicative modulation of nuclear protein kinases in the regenerating rat liver

Treatment of carboxymethylated actin with o-iodosobenzoic acid (Mahoney, W.C., and Hermodson, M.A. (1979) Biochemistry, $\text{18}$, 3810–3814) did not produce the peptide pattern expected on the basis of specific peptide bond cleavage at tryptophanyl bonds. Isolation and amino acid sequence characterization of peptides from the digest indicated that in addition to cleavage at Trp residues, cleavages occurred at Tyr-53, Tyr-198, Tyr-218, Tyr-239 and probably at Tyr-91. These results indicate that the specificity of o-iodosobenzoic acid as a reagent for peptide bond cleavage is wider than previously reported. A simple explanation for the different susceptibilities of tyrosyl-containing peptide bonds to cleavage was not apparent from inspection of the sequences adjacent to these residues.     

Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.     

Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.     

Analysis of oxygen transport in a diffusion-limited model of engineered heart tissue

Cardiac tissue engineering has made notable progress in recent years with the advent of an experimental model based on neonatal cardiomyocytes entrapped in collage gels and purified basement membrane extract, known as "engineered heart tissues" (EHTs). EHTs are a formidable display of tissue-level contractile function and cellular-level differentiation, although they suffer greatly from mass transport limitations due to the high density of metabolically active cells and the diffusion-limited nature of the hydrogel. In this report, a mathematical model was developed to predict oxygen levels inside a one-dimensional, diffusion-limited model of EHT. These predictions were then compared to values measured in corresponding experiments with a hypoxia-sensitive stain (pimonidazole). EHTs were cast between two plastic discs, which allowed for mass transfer with the culture medium to occur in only the radial direction. EHTs were cultured for up to 36 h in the presence of pimonidazole, after which time they were snap-frozen, histologically sectioned, and stained for bound pimonidazole. Quantitative image analysis was performed to measure the distance from the culture medium at which hypoxia first occurs under various conditions. As tested by variation of simple design parameters, the trends in oxygen profiles predicted by the model are in reasonable agreement with those obtained experimentally, although a number of ambiguities related to the specific model parameters led to a general overprediction of oxygen concentrations. Based on the sensitivity analysis in the present study, it is concluded that diffusion-reaction models may offer relatively precise predictions of oxygen concentrations in diffusion-limited tissue constructs.     

Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved

Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.     

Balanced expression of mitochondrial apoptosis regulatory proteins correlates with long-term survival of cardiac allografts

Abnormal regulation of apoptosis is observed in ischemic injury and may contribute to the pathogenesis of atherosclerosis. However, its role in cardiac allograft vasculopathy (CAV), the fundamental lesion of chronic rejection (CR) in heart transplantation, remains uncertain. To clarify this issue, apoptosis was quantitated in myocardium and coronary arteries from 5 cardiac allograft donors (NL) and explanted hearts of 24 patients with ischemic cardiomyopathy (IsCM) and 15 patients with CR. Tissue samples were analyzed via end-labeling fragmented DNA [via deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)] and immunoblotting for activated caspase-3 and -9. Myocyte apoptosis assessed by TUNEL was similarly increased over NL (0.21%) in both the CR (0.88%; P < 0.01) and IsCM (0.88%; P < 0.01) groups. Activated caspase-9 levels were significantly higher in CR (14.7%) compared with IsCM (6.9%; P < 0.01) and NL (0%) groups, whereas activated caspase-3 levels were similarly elevated in both CR and IsCM (7.8 and 6.5% vs. 0% in NL; P < 0.01 and P < 0.05) groups. Expression of myocardial Bcl-2 and Bax was increased in CR compared with both NL (Bax, 4.3-fold; P < 0.01; Bcl-2, 5.9-fold; P < 0.01) and IsCM (IsCM: Bax, 2.2-fold; P < 0.05; Bcl-2, 3.2-fold; P < 0.01) groups. The rate of apoptosis and the Bcl-2/Bax ratio independently correlated to graft survival in CR (activation of caspase-9: r = 0.87; P < 0.01; Bcl-2/Bax: r = 0.57; P = 0.05). Compared with native atherosclerosis, coronary arteries with CAV showed more medial apoptosis (7.8-fold; P < 0.01) and higher Bcl-2 levels (5.1-fold; P < 0.01) with lower Bax levels (threefold; P < 0.05) in the intima. These results indicate that abnormal Bcl-2 and Bax expression in myocardium and coronary arteries of cardiac allografts with CR is distinct from that in IsCM and suggest that balancing Bcl-2 to Bax in transplanted hearts promotes long-term graft survival.     

How well can blood pressure be controlled? Progress report on the Systolic Hypertension in Europe Follow-Up Study (Syst-Eur 2)

Background

The randomised, double-blind, placebo-controlled Systolic Hypertension in Europe trial (Syst-Eur 1) proved that blood pressure (BP) lowering therapy starting with nitrendipine reduces the risk of cardiovascular complications in elderly patients with isolated systolic hypertension. In an attempt to confirm the safety of long-term antihypertensive therapy based on a dihydropyridine, the Syst-Eur patients remained in open follow-up after the end of Syst-Eur 1. This paper presents the second progress report of this follow-up study (Syst-Eur 2). It describes BP control and adherence to study medications.

Methods

After the end of Syst-Eur 1 all patients, treated either actively or with placebo, were invited either to continue or to start antihypertensive treatment with the same drugs as previously used in the active treatment arm. In order to reach the target BP (sitting SBP <150 mmHg), the first line agent, nitrendipine, could be associated with enalapril and/or hydrochlorothiazide.

Results

Of the 3787 eligible patients, 3516 (93%) entered Syst-Eur 2. At the last available visit, 72% of the patients were taking nitrendipine. SBP/DBP at entry in Syst-Eur 2 averaged 160/83 mmHg in the former placebo group and 151/80 mmHg in the former active-treatment group. At the last follow-up visit SBP/DBP in the patients previously randomised to placebo or active treatment had decreased by 16/5 mmHg and 7/5 mmHg, respectively. The target BP was reached by 74% of the patients.

Conclusion

Substantial reductions in systolic BP may be achieved in older patients with isolated systolic hypertension with a treatment strategy starting with the dihydropyridine calcium-channel blocker, nitrendipine, with the possible addition of enalapril and/or hydrochlorothiazide.     

Protein-RNA interactions and virus stability as probed by the dynamics of tryptophan side chains

The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and time-resolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, time-resolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics.     

Efficiency, efficacy, and adverse effects of adenovirus vs. liposome-mediated gene therapy in cardiac allografts

Virus- and nonvirus-mediated immunosuppressive cytokine gene therapy prolongs cardiac allograft survival in various nonfunctional heart transplant animal models, but its cardiac adverse effects have not been addressed. Recently, we developed a functional heterotopic heart transplant model in rabbits. For the first time, we were able to systematically compare the efficiency, efficacy, and adverse effects of optimized adenovirus- and liposome-mediated ex vivo interleukin (IL)-10 gene transfer in functional donor hearts. The efficiency of liposome-mediated gene transfer was greatly improved in physiologically functioning donor hearts and was only three- to fourfold lower than adenovirus-mediated gene transfer. The efficacy of liposome-mediated IL-10 gene transfer was much higher than that mediated by adenovirus. Significant negative inotropic and arrhythmogenic adverse effects on transplanted hearts were observed due to viral cytotoxicity and immunogenesis, which greatly abated the therapeutic efficacy of this first generation adenovirus-mediated gene therapy.