Mice with targeted disruption of spermidine/spermine N1-acetyltransferase gene maintain nearly normal tissue polyamine homeostasis but show signs of insulin resistance upon aging

The N¹-acetylation of spermidine or spermine by spermidine/spermine N¹-acetyltransferase (SSAT) is the ratecontrolling enzymatic step in the polyamine catabolism. We have now generated SSAT knockout (SSAT-KO) mice, which confirmed our earlier results with SSAT deficient embryonic stem (ES) cells showing only slightly affected polyamine homeostasis, mainly manifested as an elevated molar ratio of spermidine to spermine in most tissues indicating the indispensability of SSAT for the spermidine backconversion. Contrary to SSAT deficient ES cells, polyamine pools in SSAT-KO mice remained almost unchanged in response to N¹, N¹¹-diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polymine pools in the wild-type animals and ES cells. Furthermore, SSATKO mice were more sensitive to the toxicity exerted by DENSPM in comparison with wild-type mice. The latter finding indicates that inducible SSAT plays an essential role in vivo in DENSPM treatmentevoked polyamine depletion, but a controversial role in toxicity of DENSPM. Surprisingly, liver polyamine pools were depleted similarly in wild type and SSAT-KO mice in response to carbon tetrachloride treatment. Further characterization of SSAT knockout mice revealed insulin resistance at old age which supported the role of polyamine catabolism in glucose metabolism detected earlier with our SSAT overexpressing mice displaying enhanced basal metabolic rate, high insulin sensitivity and improved glucose tolerance. Therefore SSAT knockout mice might serve as a novel mouse model for type 2 diabetes.     

Great tits (Parus major) searching for artificial prey: implications for cryptic coloration and symmetry

It has been suggested that bilateral symmetry may impose a costfor animals relying on camouflage because symmetric color patternsmight increase the risk of detection. We tested the effect ofsymmetry on crypsis, carrying out a predation experiment withgreat tits (Parus major) and black-and-white–patterned,artificial prey items and background. First, we found that detectiontime was significantly longer for a highly cryptic, asymmetricpattern based on a random sample of the background than forits symmetric variants. Second, we were able to arrange theelements of a prey pattern in a way that the resulting asymmetricpattern was highly cryptic and, furthermore, its symmetric variantwas highly cryptic as well. We conclude that symmetry may imposea substantial cost on cryptic patterns, but this cost variesamong patterns. This suggests that for prey, which predatorstypically view from an angle exposing their symmetry, selectionfor pattern asymmetry may be less important and selection fordecreased detectability cost of symmetry may be more importantthan previously thought. This may help to understand the existenceof so many prey with cryptic, symmetric color patterns.     

Saudis’ Attitudes toward Chemical Pollution of the Environment,Al-Baha Region,Saudi Arabia

The results of a public opinion survey were used to assess the variation in attitudes toward the chemical pollution of the environment among Saudi residents of the Al-Baha region, Saudi Arabia. A total of 17 statements were given to respondents, addressing general opinions about health risks from chemical pollution. The main findings of this study are: (1) Most Saudis consider the place where they live unhealthful; (2) there was substantial concern about exposure to chemicals with more than 77% of respondents indicating a conscious effort to avoid chemicals in their daily life; (3) chemicals were seen as predominantly being “dangerous” and leading to more harm than good, and they see that the more a person is exposed to chemicals causing cancer the more likely he/she will get cancer; (4) Respondents see that chemicals in the environment are less harmful than chemicals from lifestyle factors and they disagree with the statement that “use of chemicals has improved their health more than it has harmed them”; (5) Saudis are not prepared to accept some health risks in order to benefit the economy. The study did not find any potential relationship between gender, age range, place of residence, and education level and attitude toward chemical pollution among Saudi residents.     

Ability of short-time Fourier transform method to detect transient changes in vagal effects on hearts: a pharmacological blocking study

Conventional spectral analyses of heart rate variability (HRV) have been limited to stationary signals and have not allowed the obtainment of information during transient autonomic cardiac responses. In the present study, we evaluated the ability of the short-time Fourier transform (STFT) method to detect transient changes in vagal effects on the heart. We derived high-frequency power (HFP, 0.20-0.40 Hz) as a function of time during active orthostatic task (AOT) from the sitting to standing posture before and after selective vagal (atropine sulfate 0.04 mg/kg) and sympathetic (metoprolol 0.20 mg/kg) blockades. The HFP minimum point during the first 30 s after standing up was calculated and compared with sitting and standing values. Reactivity scores describing the fast and slow HFP responses to AOT were calculated by subtracting the minimum and standing values from the sitting value, respectively. The present results, obtained without controlled respiration, showed that in the drug-free condition, HFP decreased immediately after standing up (P < 0.001) and then gradually increased toward the level characteristic for the standing posture (P < 0.001), remaining lower than in the sitting baseline posture (P < 0.001). The magnitudes of the fast and slow HFP responses to AOT were abolished by the vagal blockade (P < 0.001) and unaffected by the sympathetic blockade. These findings indicate that HFP derived by the STFT method provided a tool for monitoring the magnitude and time course of transient changes in vagal effects on the heart without the need to interfere with normal control by using blocking drugs.     

Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.     

Generic delivery of payload of nanoparticles intracellularly via hybrid polymer capsules for bioimaging applications

Towards the goal of development of a generic nanomaterial delivery system and delivery of the 'as prepared' nanoparticles without 'further surface modification' in a generic way, we have fabricated a hybrid polymer capsule as a delivery vehicle in which nanoparticles are loaded within their cavity. To this end, a generic approach to prepare nanomaterials-loaded polyelectrolyte multilayered (PEM) capsules has been reported, where polystyrene sulfonate (PSS)/polyallylamine hydrochloride (PAH) polymer capsules were employed as nano/microreactors to synthesize variety of nanomaterials (metal nanoparticles; lanthanide doped inorganic nanoparticles; gadolinium based nanoparticles, cadmium based nanoparticles; different shapes of nanoparticles; co-loading of two types of nanoparticles) in their hollow cavity. These nanoparticles-loaded capsules were employed to demonstrate generic delivery of payload of nanoparticles intracellularly (HeLa cells), without the need of individual nanoparticle surface modification. Validation of intracellular internalization of nanoparticles-loaded capsules by HeLa cells was ascertained by confocal laser scanning microscopy. The green emission from Tb(3+) was observed after internalization of LaF(3):Tb(3+)(5%) nanoparticles-loaded capsules by HeLa cells, which suggests that nanoparticles in hybrid capsules retain their functionality within the cells. In vitro cytotoxicity studies of these nanoparticles-loaded capsules showed less/no cytotoxicity in comparison to blank capsules or untreated cells, thus offering a way of evading direct contact of nanoparticles with cells because of the presence of biocompatible polymeric shell of capsules. The proposed hybrid delivery system can be potentially developed to avoid a series of biological barriers and deliver multiple cargoes (both simultaneous and individual delivery) without the need of individual cargo design/modification.     

Multiple whole bacterial antigens in polyvalent vaccine may result in inhibition of specific responses in rainbow trout (Oncorhynchus mykiss)

The goal of fish vaccination today is to protect fish against multiple bacterial fish pathogens simultaneously using polyvalent vaccines. However, many immunological processes such as antigenic cross-reaction, antigenic competition, affinity maturation and antigen-induced suppression may affect the specificity, avidity and level of antibodies. Consequently, the biological function of antibodies may be markedly different from that predicted by conventional serologic tests. Here, we investigated the effects of vaccination and composition of vaccine on the plasma antibody levels, biological function of antibodies in opsonophagocytosis as well as the effects of vaccination on the blood leucocyte counts. Rainbow trout were vaccinated with saline or with two different polyvalent, mineral oil-adjuvanted vaccines. Vaccine 1 contained Aeromonas salmonicida, Listonella anguillarum and both Th and Fd serotypes of Flavobacterium psychrophilum antigens and vaccine 2 contained A. salmonicida, L. anguillarum and only Fd serotype of Fl. psychrophilum. The antibody-mediated opsonophagocytosis was determined as the respiratory burst (RB) activity of blood monocytes and granulocytes against the tested bacterial antigens. Three weeks after vaccination both vaccine groups and the control group showed increased RB activity against all bacterial strains. However, the increase in RB activities was non-specific and originated from the increased number of circulating granulocytes and monocytes. On the other hand, at 6 weeks post-vaccination both specific antibodies and antibody-dependent opsonophagocytosis appeared in both vaccine groups. However, the composition of the vaccine had a marked effect on the magnitude of specific responses. The Fd+Th vaccine enhanced the target specific opsonophagocytosis, to a lesser extent than the Fd vaccine. Both polyvalent vaccines appeared to mainly affect the numbers of circulating monocytes and our results suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis. Our results also suggest that the presented opsonophagocytic assay is an advantageous method to predict vaccine efficiency and that the number, and properties, of bacterial antigens in polyvalent vaccines should be carefully selected in order to avoid inhibitory effects of antigens on the specific response of fish.     

The immunohistochemical characterisation of an SV40-immortalised human corneal epithelial cell line

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.     

Spatio-temporal activation of chromatin on the human CYP24 gene promoter in the presence of 1alpha,25-Dihydroxyvitamin D3

The vitamin D3 24-hydroxylase gene (CYP24) is one of the most strongly induced genes known. Despite this, its induction by the hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3) has been characterized only partially. Therefore, we monitored the spatio-temporal, 1alpha,25OH2D3-dependent chromatin acetylation status of the human CYP24 promoter by performing chromatin immunoprecipitation (ChIP) assays with antibodies against acetylated histone 4. This was achieved by performing PCR on 25 contiguous genomic regions spanning the first 7.7 kb of the promoter. ChIP assays using antibodies against the 1alpha,25OH2D3 receptor (VDR) revealed that, in addition to the proximal promoter, three novel regions further upstream associated with VDR. Combined in silico/in vitro screening identified in three of the four promoter regions sequences resembling known VDREs and reporter gene assays confirmed the inducibility of these regions by 1alpha,25OH2D3)=. In contrast, the fourth VDR-associated promoter region did not contain any recognizable classical VDRE that could account for the presence of the protein on this region. However, re-ChIP assays monitored on all four promoter regions simultaneous association of VDR with retinoid X receptor, coactivator, mediator and RNA polymerase II proteins. These proteins showed a promoter region-specific association pattern demonstrating the complex choreography of the CYP24 gene promoter activation over 300 minutes. Thus, this study reveals new information concerning the regulation of the CYP24 gene by 1alpha,25OH2D3, and is a demonstration of the simultaneous participation of multiple, structurally diverse response elements in promoter activation in a living cell.     

Food- and temperature-dependent function of moulting rate in copepods: An example of parameterization for population dynamics models

The effects of temperature and food concentration on moultingrates in copepods were studied. The distribution of transferrates in different stages of Euterpina acuhfrons in differenttemperature and food concentrations were simulated using a processmodel. These distributions were fitted by an appropriate asymmetricalprobability distribution: the gamma density function (GDF).A parameterization of moulting rates established a continuousrelationship between parameters of GDF, and the two externalfactors, temperature and food concentration. This function canalso represent the frequency distribution of successive developmentalstages in a cohort. Two examples of such representation aregiven, using literature data.