Factors affecting yield and survival of cells when suspensions are subjected to centrifugation

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.     

Oligomerization of deoxyguanosine 5′-phosphoro-2-methylimidazolide on a polycytidylate template

The oligomerization of deoxyguanosine 5-phosphoro-2-methylimidazolide on a polycytidylate template is much less efficient than the oligomerization of the corresponding activated ribonucleotide. Nonetheless oligomers containing up to eight nucleotide residues are detected. The products are 3–5-linked oligodeoxyribonucleotides capped at the 5-terminus with a pyrophosphate-linked monomer.     

Mechanisms of beneficial effects of probucol in adriamycin cardiomyopathy

Probucol, a lipid-lowering drug, has been shown to offer protection against adriamycin-induced cardiomyopathy. In order to define the mechanism of this protection, we examined changes in antioxidants and lipid peroxidation in hearts as well as lipids in hearts and plasma from rats treated with either adriamycin or adriamycin and probucol with appropriate controls. Any potential free radical quenching as well as growth inhibitory effects of probucol were also examined using Chinese hamster ovary (CHO) cells in culture. In animal model, adriamycin caused a significant depression in glutathione peroxidase and increased plasma and cardiac lipids as well as lipid peroxidation. Probucol treatment modulated adriamycin-induced cardiomyopathic changes and increased glutathione peroxidase and superoxide dismutase activities. In the presence of adriamycin under hypoxic conditions, formation of adriamycin semiquinone radical was detected by ESR. The cell growth in these cultures was also inhibited by adriamycin in a dose-dependent manner. Probucol had no effect on adriamycin-induced growth inhibition as well as formation of semiquinone radicals. It is proposed that probucol protection against adriamycin cardiomyopathy is mediated by increased antioxidants and lipid-lowering without any effect on free radical production.     

Three-dimensional structure of an unusual Kunitz (STI) type trypsin inhibitor from Copaifera langsdorffii

The crystallographic structure of a novel trypsin inhibitor (CTI) from Copaifera langsdorffii is reported. The structure was solved by MIRAS procedure and refined to a crystallographic residual of 17.3% (R(free) = 20.3%) at 1.8 A resolution. Two isomorphous derivatives were obtained by quick cryo-soaking approach. CTI is the first structure of a member of Kunitz (STI) family formed by two noncovalently bound polypeptide chains and only one disulfide bridge. A standard Kunitz-type inhibitor has a single polypeptide chain and two disulfide bridges. Structural features granting CTI high inhibitory activity are discussed.     

Heterologous CpG island becomes extensively methylated in the genome of transgenic mice

It is demonstrated that a heterologous (chicken) CpG island containing five Sp1 canonical recognition sequences becomes highly methylated in the genome of transgenic mice bearing one or several copies of the transgene. Similar levels of methylation of the chicken CpG island were observed in different tissues of transgenic mice except the brain where the level of methylation of this chicken CpG-rich fragment was significantly lower than in other tissues. Analysis of susceptibility of the "transgenic" CpG island to Hpa II and Msp I restriction nucleases revealed an unusual methylation pattern interfering with the action of both of these enzymes. A conclusion has been drawn that heterologous CpG island per se does not contain all necessary signals permitting to maintain its own non-methylated status in the genome of transgenic animals.     

Zebrafish serotonin-N-acetyltransferase-2 gene regulation: pineal-restrictive downstream module contains a functional E-box and three photoreceptor conserved elements

Pineal function is defined by a set of very narrowly expressed genes that encode proteins required for photoperiodic transduction and rhythmic melatonin secretion. One of these proteins is serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT), which controls the daily rhythm in melatonin production. Here, pineal-specific expression of the zebrafish aanat-2 (zfaanat-2) was studied using in vivo transient expression analyses of promoter-reporter constructs; this revealed that specificity is determined by two regions located 12 kb away from each other. One is the 5'-flanking region, and the other is a 257-bp sequence, located 6 kb downstream of the transcribed region. This 3'-sequence, designated pineal-restrictive downstream module (PRDM), has a dual function: enhancement of pineal expression and inhibition of extrapineal expression. The former is an autonomic property of PRDM whereas the later function requires interaction with the upstream regulatory region of zfaanat-2. Functional analyses of the PRDM sequence revealed that three photoreceptor conserved elements (TAATC) and a single perfect E-box (CACGTG) are crucial for the dual function of PRDM. These results indicate that pineal specificity of zfaanat-2 is determined by the dual functionality of the PRDM and the interaction between upstream regulatory region and downstream photoreceptor conserved elements and E-box element.     

Cloning and developmental expression of MARK/Par-1/MELK-related protein kinase xMAK-V in<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

MAK-V/Hunk is a recently identified MARK/Par-1-related mammalian protein kinase. Although the precise function of this protein kinase is yet to be established, available data suggest its involvement in animals development and in the physiology of the nervous system. Here we report characterization of a cDNA encoding Xenopus laevis orthologue of MAK-V/Hunk protein kinase, xMAK-V. The in silico analysis also revealed MAK-V/Hunk orthologues in the fish Fugu rubripes and primitive chordate Ciona intestinalis but not in invertebrate species such as Drosophila melanogaster and Caenorhabditis elegans, suggesting that MAK-V/Hunk is a chordate-specific protein kinase. The expression of xmak-v in X. laevis embryos was analyzed using whole-mount in situ hybridization. Expression of xmak-v has been detected in all developmental stages studied including maternal expression in unfertilized eggs. The xmak-v mRNA has a predominant occurrence on the animal hemisphere of the egg, and this pattern of expression is sustained throughout cleavage and blastula stages. At the gastrula stage xmak-v expression is restricted to the ectoderm. In the later stage embryos xmak-v is expressed over the entire embryonic surface including the open neural plate at stage 15 and also in neural tube at stage 22. At tadpole stage xmak-v expression is strong in embryonic epidermis, nervous system and sensory organs, and is also obvious in perisomitic mesoderm and brachial arches.Edited by N. Satoh