Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS)

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.     

Effects of post-treatment retention interval and context on neophobia and conditioned taste aversion

We have repeatedly observed that a delay between acquisition and test, and the nature of the context in which the delay is spent, modulates latent inhibition (LI) of conditioned taste aversion (CTA; e.g. [Anim. Learn. Behav. 28 (2000) 389; Anim. Learn. Behav. 30 (2002) 112]). The present paper analysed the effects of delayed testing and treatment context after flavor exposure on the recovery of neophobia (Experiment 1) and on extinction after simple conditioning (Experiment 2). Two experiments were conducted with the same factorial design (2x2: 1 day versus 21 days of delay between first and second stage, and home versus experimental cages as place of experimental treatment). There were independent effects of both variables on habituation of neophobia and conditioning strength as measured on extinction trials. The long delay produced a reduction of neophobia (Experiment 1) and an increase in conditioning (Experiment 2). In addition, more of the flavored solution was consumed when the experimental treatment was conducted in the home cage than in the experimental cage (Experiment 1), and there was stronger conditioning when the delay period took place in the experimental cages than in the home cages (Experiment 2). The implications of these results for LI, as well as their relevance for experiments that use the CTA paradigm, are discussed.     

The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity

The SET protein and the cell cycle inhibitor p21(Cip1) interact in vivo and in vitro. We identified here the domain (157)LIF(159) of p21(Cip1) as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21(Cip1), one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21(Cip1) co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G(2)/M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21(Cip1) cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G(2)/M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G(2)/M transition by modulating cyclin B-CDK1 activity.     

Cellular and humoral responses to collagen-polyvinylpyrrolidone administered during short and long periods in humans

Collagen, particularly type I, and its related derivatives have been extensively employed in many areas of pharmacology. The present study was performed to determine the safety of collagen-polyvinylpyrrolidone (collagen-PVP) by in vitro and in vivo studies. Sera and peripheral blood cells from healthy donors without treatment and patients treated with collagen-PVP were evaluated. We observed that the biodrug does not stimulate lymphoproliferation or DNA damage in vitro, nor does it induce human anti-porcine type I collagen or anti-collagen-PVP antibodies in vivo. Furthermore, no hepatic or renal metabolic dysfunctions were observed when collagen-PVP was administered by intradermal or intramuscular routes in short- or long-term treatments. In conclusion, the present work shows that no cellular damage or immunological adverse effects (cellular and humoral) occurred during collagen-PVP treatment, even after more than 400 weeks of consecutive administrations.     

Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner

The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl(2); by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.     

Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice

The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of na?ve and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.     

Drug release from Kollicoat SR 30D-coated nonpareil beads: evaluation of coating level,plasticizer type,and curing condition

A newly available polyvinylacetate aqueous dispersion, Kollicoat SR 30D, was evaluated with respect to its ability to modulate the in vitro release of a highly water-soluble model compound (diphenhydramine hydrochloride) from nonpareil-based systems. Kollicoat SR 30D premixed with a selected plasticizer (10% wt/wt propylene glycol, 2.5% triethyl citrate, or 2.5% dibutyl sebacute), talc, and red #30 lake dye was coated onto the drug beads in an Aeromatic Strea I fluid-bed drier with a Wurster insert using bottom spray. With propylene glycol as the plasticizer, increases in polymer coating level retarded drug release from beads in a stepwise fashion along with apparent permeability, indicating a consistent release mechanisms. Stability studies at 40°C/75% RH revealed gradual decreases in dissolution rate, and additional curing studies further confirmed the dependence of release kinetics on curing condition. Furthermore, the type of plasticizer was found to play a key role. Unplasticized formulations exhibited the fastest dissolution, followed by formulations plasticized with triethyl citrate, propylene glycol, and dibutyl sebacate. All 4 formulations (unplasticized and plasticized), nevertheless, revealed a marked difference between uncured and cured dissolution profiles. Kollicoat SR 30D has, thereby, been demonstrated to effectively retard drug release from nonpareilbased systems. However, selected plasticizer type and subsequent curing condition play important roles in controlling drug release from such a system.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.