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Journal Article 2011; 32(3): 253-258 PubMed PMID: 21712771 Citation  Keywords:  Adrenal Glands:metabolism, Animals, Blotting, Western, Brain Chemistry:genetics, COS Cells, Cell Line, Cell Membrane:metabolism, Cercopithecus aethiops, Cloning, Molecular, DNA, Complementary:genetics, Immunohistochemistry, Mice, Mice, Inbred CBA, Mice, I.   

OBJECTIVE: Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal.

METHODS: VMAT-1 mRNA was detected in mouse brain with RT-PCR. The cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four different antibodies to VMAT-1.

RESULTS: Sequencing confirmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine and a proton ionophore, but not by tetrabenazine, which has a high affinity for VMAT-2. Antibodies to either the C- or N- terminus of VMAT-1 detected two proteins (73 and 55 kD) in transfected COS-1 cells. The C-terminal antibodies detected both proteins in extracts of mouse medulla/pons, cortex, hypothalamus, and cerebellum but only the 73 kD protein and higher molecular weight immunoreactive proteins in mouse adrenal and rat PC12 cells, which are positive controls for rodent VMAT-1.

CONCLUSIONS: These findings demonstrate that a functional VMAT-1 mRNA coding sequence is expressed in mouse brain and suggest processing of VMAT-1 protein differs in mouse adrenal and brain....

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Biography 2009; 30(6): 675-676 PubMed PMID: 20038930 Citation  Keywords:  Chronobiology Discipline:history, History, 20th Century, History, 21st Century, Molecular Biology:history, Neuroendocrinology:history, Romania, United States,.   

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Comparative Study 2009; 30(Suppl 1): 186-191 PubMed PMID: 20027169 Citation  Keywords:  Animals, Antioxidants:metabolism, Arvicolinae, Disease Susceptibility, Mice, Mice, Inbred BALB C, Molecular Weight, Tularemia:metabolism,.   

OBJECTIVES: Bacterium Francisella tularensis is the causative agent of tularemia disease. It is a zoonosis accompanied with high mortality when untreated. Small rodents and hares, in particular, are natural reservoirs of tularemia. Despite physiological similarity of common hosts, tularemia exerts different mortality rates. The pathogenesis of tularemia is still not fully understood. The main pathway is associated with proliferation in macrophages after activation by reactive oxygen species in phagosomes.

DESIGN: A fully virulent strain of F. tularensis subsb. holarctica was used for infection of laboratory BALB/c mice (Mus musculus) and common voles (Microtus arvalis) representing murine and microtine species. The total level of low-molecular- weight antioxidants (LMWA) in plasma was assayed by cyclic voltammetry.

RESULTS: It was found that common voles are more resistant to tularemia progression when compared to mice. When LMWA assayed, surprising changes in LMWA levels were found. Both mice and common voles were infected with high dose resulting in overall mortality. While there was a quick depletion of LMWA in plasma in mice, common voles were even able to increase LMWA.

CONCLUSION: It seems that LMWA play an important role in the organism s protection during tularemia. The ability to compensate the LMWA losses and increase levels of antioxidants in common voles is probably responsible for its lower susceptibility to tularemia....

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Journal Article 2009; 30(Suppl 1): 106-111 PubMed PMID: 20027154 Citation  Keywords:  Animals, Antigens, CD44:metabolism, Cell Line, Flow Cytometry, Hyaluronic Acid:chemistry, Lipopolysaccharides:toxicity, Macrophage Activation:drug effects, Macrophages:chemistry, Mice, Molecular Weight, NF-kappa B:metabolism, Nitric Oxide:metabolism, Nitr.   

OBJECTIVES: Hyaluronan, a linear glycosaminoglycan, is an abundant component of extracellular matrix. In its native form, the high-molar-mass hyaluronan polymers have an array of structural and regulatory, mainly anti-inflammatory and anti-angiogenic, functions. In contradiction, the biological effects of fragmented low molecular weight hyaluronan are suggested to be pro-angiogenic and pro-inflammatory.

METHODS: The effects of highly purified pharmacological grade hyaluronan of defined molecular weights 11, 52, 87, 250 and 970 kilodaltons were tested on mouse macrophage cell lines RAW 264.7 and MHS. The surface expression of CD44 and Toll-like receptor 2, surface receptors for hyaluronan, was determined by flow cytometry. Activation of macrophages was determined based on nitric oxide and tumour necrosis factor alpha production, inducible nitric oxide synthase expression, and the activation of the nuclear factor kappa B transcriptional factor.

RESULTS: Both macrophage cell lines expressed CD44 and Toll-like receptor 2, which were significantly increased by the pre-treatment of macrophages with bacterial lipopolysaccharide. Hyaluronan of any molecular weight did not activate production of nitric oxide or tumour necrosis factor alpha in any mouse macrophage cell lines. Correspondingly, hyaluronan of any tested molecular weight did not stimulate nuclear factor kappa B activation. Similarly, hyaluronan of any molecular weight neither exerted stimulatory nor inhibitory effects on macrophages pre-treated by lipopolysaccharide.

CONCLUSION: Interestingly, the data does not support the current view of low molecular weight hyaluronan as a pro-inflammatory mediator for macrophages. Further studies are necessary to clarify the effects of different molecular weight hyaluronan on phagocytes....

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Journal Article 2009; 30(Suppl 1): 72-79 PubMed PMID: 20027148 Citation  Keywords:  Absorption, Amino Acid Sequence, Animals, Apoproteins:chemistry, Butanones, Chromatography, Cytochromes b5:chemistry, Electrons, Electrophoresis, Polyacrylamide Gel, Heme:chemistry, Horses, Hydrogen-Ion Concentration, Mass Spectrometry, Molecular Sequence.   

OBJECTIVES: Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of several cytochromes P450 (CYP). To elucidate the mechanism of such modulations it is necessary to evaluate not only the effect of native cyt b5, but also that of apo-cyt b5. To prepare apo-cyt b5, heme transfer from native cyt b5 to a protein with higher affinity toward the heme, the horse heart apo-myoglobin, was utilized.

METHODS: Butanone extraction was employed to prepare apo-myoglobin. Apo-cyt b5 was separated from myoglobin by chromatography on DEAE-Sepharose. Mass spectrometry was utilized to characterize proteins eluted from DEAE- Sepharose.

RESULTS: The prepared apo-myoglobin was incubated with the cyt b5 at pH 4.2 that is the optimal pH for heme transfer from cyt b5 into apo-myoglobin. The apo-cyt b5 protein was separated from myoglobin present in the reaction mixture by chromatography on a column of DEAE-Sepharose. Using such a procedure, 16% yield of apo-cyt b5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b5. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native cyt b5. The prepared apo-cyt b5 reconstituted with heme can receive electrons from NADPH:CYP reductase.

CONCLUSION: A biologically active apo-cyt b5 was prepared using transfer of heme from cyt b5 to horse heart apo-myoglobin by the procedure described here....

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Journal Article 2009; 30(4): 525-529 PubMed PMID: 20010486 Citation  Keywords:  Animals, Base Sequence, Female, Molecular Sequence Data, Osteopontin:genetics, Ovary:physiology, Oviducts:physiology, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Promoter Regions, Genetic:genetics, Reproduction:genetics, Reverse .   

OBJECTIVES: The aim of the present study was to find a possible polymorphism in the promoter region of the osteopontin (OPN) gene, as a potential mutation region, connected with the transcription factor-binding sites or regulatory sequences and to estimate the expression of this gene in ovaries and oviduct of sows.

MATERIAL AND METHODS: Sixty wbp x pbz sows after the first mating were slaughtered and tissues samples of the ovaries and oviduct were taken. Primer pairs for PCR analysis were designed on the basis of swine osteopontin 5' end sequence driven from GenBank. This study's amplified DNA fragment, spanning 274 bp of the promoter region, was chosen because of its contents of tree specific sites: type II collagen silence sequence (CACCTCC) at -682 (from the transcription initiation site), glucocortIcoid response site (TGTCCT) at -658 and CAAT box -592. This DNA fragment was subjected to a MSSCP analysis.

RESULTS: A different MSSCP pattern was shown which indicates that mutation is located in this region. The samples of different conformers were sequenced and the A --> G transitions was identified in two positions -617 and -608. Restriction analysis of the DNA was performed, but unfortunately none of the known restriction enzymes recognized the novel SNPs, which is why the specific primer pairs characteristic for nucleotide A or for nucleotide G were chosen. In the second stage of the presented study the total RNA was extracted from the tissues of the ovaries and oviduct and the complementary DNA (cDNA) was synthesized.

CONCLUSION: The real-time PCR analysis to determined the expression dynamics of the OPN gene in pig tissues was performed....

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Journal Article 2008; 29(5): 697-701 PubMed PMID: 18987579 Citation  Keywords:  Antioxidants:chemistry, Ascorbic Acid:chemistry, Carbohydrate Sequence, Copper:chemistry, Free Radicals:chemistry, Hyaluronic Acid:chemistry, Hydrogen Peroxide:chemistry, Hydroxyl Radical:chemistry, Kinetics, Molecular Weight, Oxidants:chemistry, Penicill.   

OBJECTIVES: The study presents results of pro- and anti-oxidative effects of D-penicillamine on hyaluronan degradation by ascorbate plus cupric ions.

METHODS: The well established degradative system comprising high-molar-mass hyaluronan and ascorbate plus Cu(II) ions was used. Primarily, the effects of replacement of ascorbic acid in this system by D-penicillamine were investigated. Then, D-penicillamine was added into the above degradative system before reaction onset or 1h after the reaction had started. To monitor hyaluronan degradation kinetics, rotational viscometry was applied.

RESULTS: No hyaluronan degradation occurred when ascorbate was replaced by D-penicillamine. The drug addition into the complete degradative system at the reaction onset caused a marked inhibition of hyaluronan degradation. However, the inhibitory effect turned to a pro-oxidative one within appr. 1 h.

CONCLUSION: The dual behavior of D-penicillamine on hyaluronan degradation can relate to: (i) the drug completely traps *OH radicals generated from ascorbate plus Cu(II) ions under aerobic conditions; (ii) thiyl radicals generated from D-penicillamine react with D-penicillamine anions resulting in novel radical reactive species, which e.g. by reducing dioxygen molecules can generate further *OH radicals....

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Journal Article 2008; 29(4): 391-398 PubMed PMID: 18766165 Citation  Keywords:  Animals, Antioxidants:metabolism, Free Radicals:metabolism, Melatonin:metabolism, Mitochondria:metabolism, Molecular Structure, Neuroprotective Agents:metabolism, Oxidative Stress, Reactive Nitrogen Species:metabolism, Reactive Oxygen Species:metabolism,.   

: N-acetyl-5-methoxytryptamine (melatonin) is an endogenous indoleamine produced by all vertebrate organisms. Its production in the pineal gland has been extensively investigated but other organs also synthesize this important amine. Melatonin's functions in organisms are diverse. The actions considered in the current review relate to its ability to function in the reduction of oxidative stress, i.e., molecular damage produced by reactive oxygen and reactive nitrogen species. Numerous publications have now shown that not only is melatonin itself an efficient scavenger of free radicals and related reactants, but so are its by-products cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, and others. These derivatives are produced sequentially when each functions in the capacity of a free radical scavenger. These successive reactions are referred to as the antioxidant cascade of melatonin. That melatonin has this function within cells has been observed in studies employing time lapse conventional, confocal and multiphoton fluorescent microscopy coupled with the use of appropriate mitochondrial-targeted fluorescent probes. The benefits of melatonin and its metabolites have been described in the brain where they are found to be protective in models of Parkinson's disease, Alzheimer's disease and spinal cord injury. The reader is reminded, however, that data not covered in this review has documented beneficial actions of these amines in every organ where they have been tested. The outlook for the use of melatonin in clinical trials looks encouraging given its low toxicity and high efficacy....

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