Effect of various chloride salts on the utilization of phosphorus, calcium, and magnesium

Only part of the effect of dietary protein on urinary calcium excretion can be ascribed to sulfur amino acids. We hypothesized that chloride, another factor often associated with isolated proteins, and another amino acid, lysine, affect utilization of calcium. The effects of supplemental dietary chloride, inorganic or organic, on calcium, phosphorus, and magnesium utilization were studied in two rat studies. Weanling Sprague-Dawley rats were fed semi-purified diets that contained moderate (1.8 mg Cl/g diet) or supplemental (15.5 mg Cl/g diet) chloride as sodium chloride, potassium chloride, or lysine monohydrochloride with or without calcium carbonate for 56 or 119 days. Rats fed supplemental sodium chloride or potassium chloride had higher urinary phosphorus excretion, more efficient phosphorus absorption, but unchanged tissue phosphorus levels after 7 and 16 weeks of dietary treatment as compared to rats fed moderate chloride. Rats fed supplemental sodium chloride or potassium chloride excreted more calcium in urine at 7 weeks and absorbed calcium less efficiently at 16 weeks. Tissue calcium concentrations were unaffected, but total tibia magnesium and plasma magnesium concentrations were lower in rats fed supplemental sodium chloride or potassium chloride than those fed moderate chloride. Lysine chloride with or without additional calcium elevated urinary calcium excretion even more than sodium chloride and potassium chloride ingestion. Rats fed lysine chloride with supplemental calcium had smaller apparent absorption and urinary losses of phosphorus and magnesium after 16 weeks and lower tibia and plasma magnesium concentrations than rats fed lysine chloride.     

Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Studies of mitochondrial respiration in muscle cells in situ: Use and misuse of experimental evidence in mathematical modelling

Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.     

Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle

Recent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered--localization of diffusion restrictions--is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells.     

Ancient metalloenzymes as possible markers in molecular archaeology

The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology.     

Study of possible interactions of tubulin, microtubular network, and STOP protein with mitochondria in muscle cells

We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 μM tubulin heterodimer increased apparent K _m for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K _m for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the β-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process.     

The extraction,structure, and gelling properties of hybrid galactan from the red alga <Emphasis Type="Italic">Furcellaria lumbricalis</Emphasis> (Baltic Sea,Estonia)

The structure and composition of galactan from Furcellaria lumbricalis (furcellaran) were investigated in connection with rheological specificities, gel structure, and extraction conditions. The polysaccharide was characterized by ¹³C nuclear magnetic resonance (¹³C-NMR) and Fourier transform infrared spectroscopy, inductively coupled plasma-optical emission spectrometry, electrothermal atomization atomic absorption spectrometry, and gel permeation chromatography methods. The microstructure of polymer gels was studied using a cryofixation method in combination with freeze-drying and scanning electron microscopy (SEM) techniques. The undersulfated furcellaran backbone consists mainly of 3,6-anhydro-d-galactose (28.5–30.1%) and galactose residues, the latter being partly sulfated in positions 4 and 6, which give rise to some specific properties of the gel. Also, residues of 6-O-methyl-d-galactose as a minor component are found to be present. The water-extracted furcellaran with the average molecular weight about 290 kDa is rich in nitrogen, calcium, magnesium, and potassium, while the sodium content is rather low. The low sulfur content (5.3%) and ¹³C-NMR spectra refer to an undersulfated nature of this galactan. The extraction of seaweeds in low concentration alkaline solutions (instead of water) leads to a significant increase of the minimum size of the galactan particles and the value of gel strength (more than 12 times for Rb-containing gels). The properties of the gel are dependent on specific tentacle-like structure units present in furcellaran gels established by a high-resolution SEM.     

Alopecia areata in a rhesus monkey (Macaca mulatta)

BACKGROUND: A 14-year-old female rhesus monkey (Macaca mulatta) of Chinese origin has been suffering from alopecia universalis since childhood. METHODS: Recently, the health status of the animal was recorded comprehensively by detailed clinical examination including hematology and serology supplemented by histological and immunohistochemical investigations of skin biopsies and molecular biological techniques to clarify the causes of the persistent hair loss. RESULTS AND CONCLUSIONS: The hairless gene (hr) nonsense mutation was ruled out by polymerase chain reaction and by sequencing of the corresponding gene. Histological examinations revealed a prominent chronic lymphocytic perifolliculitis and folliculitis affecting anagen stage hair follicles as well as miniaturized hair follicles. Immunohistochemistry using the antibodies CD3, CD20 and CD4 confirmed the diagnosis of a T-cell-mediated autoimmune disease resembling alopecia areata universalis in humans.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.