Dose-dependent effects of leucine supplementation on preservation of muscle mass in cancer cachectic mice.

Cancer cachexia, which is characterized by muscle wasting, is associated with increased morbidity and mortality. Because muscle protein synthesis may be increased and protein breakdown reduced by leucine supplementation, we used the C26 tumor-bearing cachectic mouse model to assess the effects of dietary supplementation with leucine on muscle weight and the markers of muscle protein breakdown (mRNA of atrogin and murf). Male CD2F1 mice were subcutaneously inoculated with tumor cells (tumor-bearing mice; TB) or were sham injected (control; C). They were fed standard diets or diets supplemented with leucine [1 gr (TB1Leu) or 8 gr (TB8Leu) supplemented leucine per kg feed]; TB and C received 8.7% Leu/g protein, TB1Leu received 9.6% Leu/g protein and TB8Leu received 14.6 Leu/g protein. After 21 days, the following were determined: body weights, plasma amino-acid concentrations, tumor size and muscle mass of the gastrocnemius (mG), tibialis anterior (mTA), extensor digitorum longus (mEDL) and soleus (mS) muscles. In tumor-bearing (TB) mice, carcass and skeletal muscle masses decreased, and levels of atrogin and murf mRNA in the mEDL increased. Muscle-mass loss was counteracted dose-dependently by leucine supplementation: relative to TB, the mass of the mG was +23% in TB8Leu, and +22% in mTA (p<0.05). However, leucine supplementation did not change atrogin and murf mRNA levels. Total plasma amino acid concentrations increased in TB, especially for taurine, lysine, arginine and alanine (p<0.05). Leucine supplementation attenuated the increase in total plasma amino-acid concentrations (p<0.05). Irrespective of changes in muscle protein breakdown markers, leucine supplementation reduced muscle wasting in tumor-bearing cachectic mice and attenuated changes in plasma amino acids.

Dietary supplementation with a specific combination of high protein, leucine, and fish oil improves muscle function and daily activity in tumour-bearing cachectic mice.

Cancer cachexia is characterised by metabolic alterations leading to loss of adipose tissue and lean body mass and directly compromises physical performance and the quality of life of cancer patients. In a murine cancer cachectic model, the effects of dietary supplementation with a specific combination of high protein, leucine and fish oil on weight loss, muscle function and physical activity were investigated. Male CD2F1 mice, 6-7 weeks old, were divided into body weight-matched groups: (1) control, (2) tumour-bearing, and (3) tumour-bearing receiving experimental diets. Tumours were induced by s.c. inoculation with murine colon adenocarcinoma (C26) cells. Food intake, body mass, tumour size and 24 h-activity were monitored. Then, 20 days after tumour/vehicle inoculation, the animals were killed and muscle function was tested ex vivo. Tumour-bearing mice showed reduced carcass, muscle and fat mass compared with controls. EDL muscle performance and total daily activity were impaired in the tumour-bearing mice. Addition of single nutrients resulted in no or modest effects. However, supplementation of the diet with the all-in combination of high protein, leucine and fish oil significantly reduced loss of carcass, muscle and fat mass (loss in mass 45, 52 and 65% of TB-con, respectively (P<0.02)) and improved muscle performance (loss of max force reduced to 55-64% of TB-con (P<0.05)). Moreover, total daily activity normalised after intervention with the specific nutritional combination (50% of the reduction in activity of TB-con (P<0.05)). In conclusion, a nutritional combination of high protein, leucine and fish oil reduced cachectic symptoms and improved functional performance in cancer cachectic mice. Comparison of the nutritional combination with its individual modules revealed additive effects of the single components provided.

Direct effects of doxorubicin on skeletal muscle contribute to fatigue.

Chemotherapy-induced fatigue is a multidimensional symptom. Oxidative stress has been proposed as a working mechanism for anthracycline-induced cardiotoxicity. In this study, doxorubicin (DOX) was tested on skeletal muscle function. Doxorubicin induced impaired ex vivo skeletal muscle relaxation followed in time by contraction impediment, which could be explained by DOX-induced changes in Ca(2+) responses of myotubes in vitro. The Ca(2+) responses in skeletal muscle, however, could not be explained by oxidative stress.

Beneficial immune modulatory effects of a specific nutritional combination in a murine model for cancer cachexia.

The majority of patients with advanced cancer are recognised by impaired immune competence influenced by several factors, including the type and stage of the tumour and the presence of cachexia. Recently, a specific nutritional combination containing fish oil, specific oligosaccharide mixture, high protein content and leucine has been developed aimed to support the immune system of cancer patients in order to reduce the frequency and severity of (infectious) complications. In a recently modified animal model cachexia is induced by inoculation of C26 tumour cells in mice. In a pre-cachectic state, no effect was observed on contact hypersensitivity, a validated in vivo method to measure Th1-mediated immune function, after adding the individual nutritional ingredients to the diet of tumour-bearing mice. However, the complete mixture resulted in significantly improved Th1 immunity. Moreover, in a cachectic state, the complete mixture reduced plasma levels of pro-inflammatory cytokines and beneficially affected ex vivo immune function. Accordingly, the combination of the nutritional ingredients is required to obtain a synergistic effect, leading to a reduced inflammatory state and improved immune competence. From this, it can be concluded that the specific nutritional combination has potential as immune-supporting nutritional intervention to reduce the risk of (infectious) complications in cancer patients.

[18F]FHPG positron emission tomography for detection of herpes simplex virus (HSV) in experimental HSV encephalitis.

Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial. Subtle brain infections with HSV may be causally related to neuropsychiatric disorders such as Alzheimer's dementia. We investigated the feasibility of a noninvasive positron emission tomography (PET) imaging technique using [(18)F]FHPG as a tracer for the detection of HSE. For this purpose, rats received HSV-1 (infected group) or phosphate-buffered saline (control group) by intranasal application, and dynamic PET scans were acquired. In addition, the distribution of tracer accumulation in specific brain areas was studied with phosphor storage imaging. The PET images revealed that the overall brain uptake of [(18)F]FHPG was significantly higher for the infected group than for control animals. Phosphor storage images showed an enhanced accumulation of [(18)F]FHPG in regions known to be affected after intranasal infection with HSV. High-performance liquid chromatography metabolite analysis showed phosphorylated metabolites of [(18)F]FHPG in infected brains, proving that the increased [(18)F]FHPG uptake in infected brains was due to HSV thymidine kinase-mediated trapping. Freeze lesion experiments showed that damage to the blood-brain barrier could in principle induce elevated [(18)F]FHPG uptake, but this nonspecific tracer uptake could easily be discriminated from HSE-derived uptake by differences in the tracer kinetics. Our results show that [(18)F]FHPG PET is a promising tool for the detection of HSV encephalitis.

The expression of the neuronal intermediate filament protein peripherin in the rat embryo.

The expression of the neuronal type III intermediate filament protein peripherin was examined in the rat embryo during and following neuronogenesis in the spinal cord and the peripheral nervous system. In situ hybridization analysis reveals that peripherin mRNA is found in the mid-gestational rat embryo in ventral and lateral motoneurons in the spinal cord, and in neurons of all peripheral ganglia examined, including spinal, sympathetic, and enteric ganglia. Peripherin mRNA is seen only in post-migratory motoneurons or neuronal cells in aggregating ganglia, indicating that precursor cells do not express peripherin. To examine the expression of the protein, an affinity-purified antibody (anti-per) specific for a bacterially produced peripherin fusion protein was generated. Anti-per specifically recognizes a 58 kDa, cytoskeletal-enriched, nerve growth factor (NGF)-inducible protein of the expected tissue distribution. Immunocytodetection with anti-per shows that the initiation of peripherin protein synthesis is coincident with the morphological differentiation of neurons. In development, peripherin is one constituent of a program of gene expression activated at terminal neuronal differentiation.

Chromosomal mapping and nucleotide sequence of a human DNA autonomously replicating sequence.

A 1.1-kb human DNA fragment (ARSH1) capable of functioning as a putative origin of replication in yeast cells has been characterized both by in situ hybridization to human metaphase chromosomes and by DNA sequencing. Our hybridization studies show a preferential localization of ARSH1 in chromosome regions 1p34-36 and 2q34-37. DNA sequence analysis indicates that in addition to the consensus sequence required for ARS function in yeast cells, nuclear matrix-associated DNA motifs are also present in the 1.1-kb fragment. These results suggest that ARSH1 sequences may serve as points of anchorage to the nuclear matrix for chromosomes 1 and 2.

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation.

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.