Regeneration of cryoresistance of in vitro rumen ciliate cultures

The purpose of this study was to investigate factors affecting mechanical- and cryo-resistance of the rumen ciliates Entodinium caudatum (E.c.), Entodinium furca monolobum (E.f.m.), Entodinium simplex (E.s.), Diplodinium denticulatum (two clones, D.d.01 and D.d.02), Diploplastron affine (D.a.) and Epidinium ecaudatum forma caudatum (E.e.c.) after long-term in vitro cultivation. Following prolonged in vitro cultivation (more than six months), the ciliates were very sensitive to both centrifugation and 5% (v/v) dimethylsulphoxide, with motility decreased to: 39 and 23% for E.c., 66 and 32% for E.f.m., 46 and 27% for D.d. 01, 64 and 41% for D.a., and 44 and 28% for E.e.c., respectively. Thus, cryopreservation was unsuccessful. The effect of supplementing the ciliate growth medium with rumen fluid, glycine-betaine, proline, myo-inositol, linoleic acid, Sel-Plex or insulin, together with the effect of the source of rumen fluid on ciliate resistance to centrifugation, dimethylsulphoxide and freezing was also tested. The omission of rumen fluid from the growth medium resulted in the loss of cryoresistance after one-month cultivation. Supplementing the growth environment with a combination of glycine-betaine, proline, linoleic acid, Sel-Plex, insulin plus improved quality rumen fluid significantly enhanced survival of the ciliates after the freezing-thawing procedure (from 1 to 33% survival in un-supplemented vs. supplemented for E.c., P < 0.01; 4-40% E.f.m., P < 0.01; 0-17% D.d., P < 0.05; 5-7% D.a. and 4-36% E.e.c., P < 0.01).     

Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis

Agrobacterium-mediated transient assays for gene function are increasingly being used as alternatives to genetic complementation and stable transformation. However, such assays are variable and not equally successful in different plant species. We analysed a range of genetic and physiological factors affecting transient expression following agroinfiltration, and developed a protocol for efficient and routine transient assays in several plant species. Lettuce exhibited high levels of transient expression and was at least as easy to work with as Nicotiana benthamiana. Transient expression occurred in the majority of cells within the infiltrated tissue and approached 100% in some regions. High levels of transient expression were obtained in some ecotypes of Arabidopsis; however, Arabidopsis remains recalcitrant to routine, genotype-independent transient assays. Transient expression levels often exceeded those observed in stably transformed plants. The laboratory Agrobacterium tumefaciens strain C58C1 was the best strain for use in plant species that did not elicit a necrotic response to A. tumefaciens. A wild A. tumefaciens strain, 1D1246, was identified that provided high levels of transient expression in solanaceous plants without background necrosis, enabling routine transient assays in these species.     

Expression of cell survival/death genes: Bcl-2 and Bax at the rate of colon cancer prognosis

The rate of tumour growth is dependent on the balance between proliferation and apoptosis at all stages of carcinogenesis. Apoptosis inhibition, in turn, depends partly on the balance between expression of two cell death regulatory genes, Bcl-2 and Bax. Colon cancer has long been associated with disturbances in apoptosis regulation. The aim of our study was to determine the expression levels of Bcl-2 and Bax mRNAs in 1 microg sample of total RNA obtained from normal colon and colon adenocarcinoma. This study was intended to evaluate possible differences in Bcl-2 and Bax mRNA levels at particular stages of colon adenocarcinoma classified according to Duke's system. The apoptotic frequency (represented by Bax mRNA copy number) was inversely proportional to the decrease of Bcl-2 gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to confirm apoptosis.     

Endothelial NADH/NADPH-dependent enzymatic sources of superoxide production: relationship to endothelial dysfunction

There is growing evidence that endothelial dysfunction, which is often defined as the decreased endothelial-derived nitric oxide (NO) bioavailability, is a crucial factor leading to vascular disease states such as hypertension, diabetes, atherosclerosis, heart failure and cigarette smoking. This is due to the fact that the lack of NO in endothelium-dependent vascular disorders contributes to impaired vascular relaxation, platelet aggregation, increased vascular smooth muscle proliferation, and enhanced leukocyte adhesion to the endothelium. During the last several years, it has become clear that reduction of NO bioavailability in the endothelium-impaired function disorders is associated with an increase in endothelial production of superoxide (O(2)(*-)). Because O(2)(*-) rapidly scavenges NO within the endothelium, a reduction of bioactive NO might occur despite an increased NO generation. Among many enzymatic systems that are capable of producing O(2)(*-), NAD(P)H oxidase and uncoupled endothelial NO synthase (eNOS) apparently are the main sources of O(2)(*-) in the endothelial cells. It seems that O(2)(*-) generated by NAD(P)H oxidase may trigger eNOS uncoupling and contribute to the endothelial balance between NO and O(2)(*-). That is maintained at diverse levels.     

Ubc9-induced inhibition of diadenosine triphosphate hydrolase activity of the putative tumor suppressor protein Fhit

Fhit protein is the product of the putative tumor suppressor fragile histidine triad (FHIT) gene. The way by which Fhit exerts its antitumor activity remains largely unknown, although the Fhit-Ap3A complex is believed to be the native signaling form of Fhit. Here, we have shown that Fhit protein interacts with hUbc9, a recombinant human SUMO-1 conjugating enzyme, in an adenosine(5')triphospho(5')nucleoside (Ap3N)-dependent manner. Our experiments showed that the dinucleoside polyphosphate hydrolase activity of Fhit is suppressed by interacting with hUbc9 protein. In the presence of equimolar hUbc9 the Vmax and Km activity of Fhit was decreased by 35%. Analysis of Fhit kinetics in the presence of different fixed concentrations of Ubc9 showed that Ubc9 is an uncompetitive inhibitor. Including SUMO-1 protein in the assay neither affected the Fhit activity nor modified the effect of Ubc9 on Fhit kinetics. Our data suggest that hUbc9-induced inhibition of Fhit may result in an elongation of the Fhit-Ap3A signaling complex lifetime leading to alteration of its antitumor activity.     

Choice of isolation procedure for endotoxins from the outer membrane of the bacteria Desulfovibrio desulfuricans

The chemical structure determining properties and biological functions of endotoxins derived from Desulfovibrio desulfuricans species has not been recognized, which considerably hinders the choice of an effective extraction procedure of these lipopolysaccharides (LPS) from the bacterial outer cell membrane. We aimed at selecting the most effective method of LPS isolation from D. desulfuricans in terms of the most efficient extraction solution, the appropriate conditions of isolation and adequate purification technique. For this purpose we tested a few literature-based procedures utilizing various extraction mixtures (phenol-water, phenol-chloroform-petroleum ether and Tri-Reagent, i.e. aqueous solution of guanidinum thiocyanate and phenol). The best yield and purity of the isolated LPS were provided by the application of the extraction with phenol-water according to the modified by Shnyra et.al. (2000) procedure of Westphal et. al. (1952). A satisfactory method of isolation in micro scale appeared to be that based on Tri-Reagent and propagated by Yi and Hackett in 2000. The extraction of LPS from D. desulfuricans with phenol-chloroform-petroleum ether should not be recommended due to its low efficiency.     

Structural basis for budding by the ESCRT-III factor CHMP3

The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Estimation of arterial and cardiopulmonary total peripheral resistance baroreflex gain values: validation by chronic arterial baroreceptor denervation

Feedback control of total peripheral resistance (TPR) by the arterial and cardiopulmonary baroreflex systems is an important mechanism for short-term blood pressure regulation. Existing methods for measuring this TPR baroreflex mechanism typically aim to quantify only the gain value of one baroreflex system as it operates in open-loop conditions. As a result, the normal, integrated functioning of the arterial and cardiopulmonary baroreflex control of TPR remains to be fully elucidated. To this end, the laboratory of Mukkamala et al. (Mukkamala R, Toska K, and Cohen RJ. Am J Physiol Heart Circ Physiol 284: H947-H959, 2003) previously proposed a potentially noninvasive technique for estimating the closed-loop (dimensionless) gain values of the arterial TPR baroreflex (GA) and the cardiopulmonary TPR baroreflex (GC) by mathematical analysis of the subtle, beat-to-beat fluctuations in arterial blood pressure, cardiac output, and stroke volume. Here, we review the technique with additional details and describe its experimental evaluation with respect to spontaneous hemodynamic variability measured from seven conscious dogs, before and after chronic arterial baroreceptor denervation. The technique was able to correctly predict the group-average changes in GA and GC that have previously been shown to occur following chronic arterial baroreceptor denervation. That is, reflex control by the arterial TPR baroreflex was virtually abolished (GA = -2.1 +/- 0.6 to 0.3 +/- 0.2; P < 0.05), while reflex control by the cardiopulmonary TPR baroreflex more than doubled (GC = -0.7 +/- 0.4 to -1.8 +/- 0.2; P < 0.05). With further successful experimental testing, the technique may ultimately be employed to advance the basic understanding of TPR baroreflex functioning in both humans and animals in health and disease.     

The physical and chemical properties of eumelanin

In this article, we review the current state of knowledge concerning the physical and chemical properties of the eumelanin pigment. We examine properties related to its photoprotective functionality, and draw the crucial link between fundamental molecular structure and observable macroscopic behaviour. Where necessary, we also briefly review certain aspects of the pheomelanin literature to draw relevant comparison. A full understanding of melanin function, and indeed its role in retarding or promoting the disease state, can only be obtained through a full mapping of key structure-property relationships in the main pigment types. We are engaged in such an endeavor for the case of eumelanin.