Cryptosporidium parvum infection in cattle: are current perceptions accurate?

The perception that cattle are major reservoirs for Cryptosporidium parvum infections in humans and that C. parvum is a major cause of diarrhea and production loss in cattle might not reflect the whole situation. Numerous management factors influence the epidemiological and clinical picture associated with C. parvum infections in cattle. Whereas C. parvum is highly prevalent in young dairy calves and confined beef calves, it occurs rarely in calves on range and in adult cattle. In well-managed herds, clinical disease due to C. parvum is also rare. Therefore, C. parvum infections in cattle might not be as important as current perceptions would indicate.     

Fast cadmium inhibition of photosynthesis in cyanobacteria in vivo and in vitro studies using perturbed angular correlation of γ-rays

The effect of cadmium on the photosynthetic activity of Synechocystis PCC 6803 was monitored in this study. The oxygen evolving capacity of Synechocystis treated with 40 μM CdCl₂ was depressed to 10% of the maximum in 15 min, indicating that Cd²⁺ penetrated rapidly into the cells and blocked the photosynthetic activity. However, neither photosystem II (PSII) nor photosystem I (PSI) activity showed a significant short-term decrease which would explain this fast decrease in the whole-chain electron transport. Thermoluminescence measurements have shown that the charge separation and stabilization in PSII remains essentially unchanged during the first few hours following the Cd²⁺ treatment. The electron flow through PSI was monitored by following the redox changes of the P700 reaction centers of PSI. Alterations in the oxidation kinetics of P700 in the Cd²⁺-treated cells indicated that Cd²⁺ treatment might affect the available electron acceptor pool of P700, including the CO₂ reduction and accumulation in the cells. Perturbed angular correlation of γ-rays (PAC) using the radioactive ^111mCd isotope was used to follow the Cd²⁺ uptake at a molecular level. The most plausible interpretation of the PAC data is that Cd²⁺ is taken up by one or more Zn proteins replacing Zn²⁺ in Synechocystis PCC 6803. Using the radioactive ¹⁰⁹Cd isotope, a protein of approximately 30 kDa that binds Cd²⁺ could be observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicate that Cd²⁺ might inactivate different metal-containing enzymes, including carbonic anhydrase, by replacing the zinc ion, which would explain the rapid and almost full inhibition of the photosynthetic activity in cyanobacteria.     

Intra-isolate Heterogeneity and Reproducibility of PCR-based Genotyping of Cryptosporidium parvum Using the β-tubulin Gene

Cryptosporidium parvum is a common contaminant in surface waters and presents significant problems for the water industry, public health and agriculture. Consequently, ascertaining the contaminating source of waterborne oocysts is of paramount importance. Based on currently available information, isolates of C. parvum can be differentiated into at least two genotypes using polymorphic genetic markers: genotype 1, to date isolated almost exclusively from humans, and genotype 2 isolates from humans and many other animals. Differentiation into these two genotypes has been based on either restriction fragment length polymorphisms or sequencing of PCR amplified gene fragments. The objective of this study was to evaluate the reproducibility of genotyping methods using a single isolate of C. parvum. A 620 bp fragment of the C. parvum -tubulin gene, generated by PCR from multiple aliquots of a single preparation of oocysts of the Iowa isolate, was sequenced. Significant sequence heterogeneity was detected within this single isolate; there was more sequence variation between clones originating from the Iowa isolate (up to 0.9 %) than between individual clones originating from different isolates of C. parvum. Over 6 % of the -tubulin gene sequence positions (38 out of 620 bp) were variable when comparing multiple clones from the one isolate. The results indicated that while the various procedures used for genotyping isolates may introduce some sequence errors, the Iowa isolate used for this investigation appeared to be composed of multiple sub-genotypes. While none of the sequence variations resulted in clones of the Iowa isolate (genotype 2) being mis-identified as genotype 1, the results have important implications if minor sequence variations are to be used for subtyping isolates and drawing conclusions regarding the origin of, or relationships between, C. parvum oocysts in water and the community.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Effects of γ-irradiation on antioxidant activity in soybean seeds

There are some reports that low doses of γ-irradiation could induce antioxidant activities in plant material, including soybean. Irradiation, required for the inactivation of some pathogens and induction of mutations, may have adverse effects on sensorial, nutritional and antioxidant qualities. The effects of different γ-irradiation doses (100–200 Gy) on antioxidant properties of soybean seeds was investigated. In this study, we report the results obtained by analysis of antioxidant enzyme activities, reduced glutathione, malonyldialdehyde (MDA) and hydroxyl (HO⁻) radical quantities, soluble protein content, and total antioxidant activity in irradiated soybean seeds. Antioxidant enzyme activities were affected due to high irradiation intensity. Significant changes of total antioxidant activity and MDA and HO.quantities were observed only under the highest irradiation dose, with a 15.7% reduction in total antioxidant activity, MDA quantity increase of 21.6%, and HO⁻ radical quantity increase of 79.3% compared to the non-irradiated control. The total soluble protein content increased slightly.     

Localization of lipid peroxidation products in liposomes after γ-irradiation

The distribution of lipid peroxidation products in liposomes after γ-irradiation at various doses was studied. Increases in thiobarbituric-acid-reactive substances, in the absorbance at 232 nm and in hydroperoxides were observed mainly in liposomal membranes after relatively low doses of irradiation, while carbonyl compounds were distributed both inside and outside the membranes. After higher doses of irradiation, however, the absorbance at 232 nm and the amount of hydroperoxides reached a maximal level in the membrane portion and then decreased when the decomposition products were released from the membranes. Under this condition, malondialdehyde and other carbonyl compounds were increased mainly in the medium of liposomal suspension. These results are discussed with reference to the lipid peroxidation process which is induced quantitatively by ionizing radiation.     

Increased expression of PS1 is sufficient to elevate the level and activity of γ-secretase in vivo

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.     

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies

BACKGROUND:

Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression.

MATERIALS AND METHODS:

A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously.

RESULTS:

A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro.

CONCLUSION:

Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.     

Lack of PPARγ in myeloid cells confers resistance to Listeria monocytogenes infection

The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγ(flox/flox)). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6C(hi) monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.     

ATM is required for rapid degradation of cyclin D1 in response to γ-irradiation

The cellular response to DNA damage induced by γ-irradiation activates cell-cycle arrest to permit DNA repair and to prevent replication. Cyclin D1 is the key molecule for transition between the G1 and S phases of the cell-cycle, and amplification or overexpression of cyclin D1 plays pivotal roles in the development of several human cancers. To study the regulation of cyclin D1 in the DNA-damaged condition, we analyzed the proteolytic regulation of cyclin D1 expression upon γ-irradiation. Upon γ-irradiation, a rapid reduction in cyclin D1 levels was observed prior to p53 stabilization, indicating that the stability of cyclin D1 is controlled in a p53-independent manner. Further analysis revealed that irradiation facilitated ubiquitination of cyclin D1 and that a proteasome inhibitor blocked cyclin D1 degradation under the same conditions. Interestingly, after mutation of threonine residue 286 of cyclin D1, which is reported to be the GSK-3β phosphorylation site, the mutant protein showed resistance to irradiation-induced proteolysis although inhibitors of GSK-3β failed to prevent cyclin D1 degradation. Rather, ATM inhibition markedly prevented cyclin D1 degradation induced by γ-irradiation. Our data indicate that communication between ATM and cyclin D1 may be required for maintenance of genomic integrity achieved by rapid arrest of the cell-cycle, and that disruption of this crosstalk may increase susceptibility to cancer.     

Effect of γ-irradiation on F-2 and T-2 toxin production in corn and rice

Fusarium graminearum andF. tricinctum were grown on moistened corn and rice. After inoculation the substrates were exposed to γ-irradiation and growth rate together with mycotoxin production were measured. A delay in mycelium growth and an increase in F-2 and T-2 toxin production occurred after irradiation with 1 and 3 kGy. The maximum F-2 production was 10.7 mg/kg on rice at 3 kGy, whereas T-2 was 735 μg/kg on rice at 3 kGy. At 9 kGy neither growth nor toxin production could be detected in any inoculated corn and rice substrate.     

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic γ-irradiation

Summary Lymphoblastoid cell lines (LCLs) derived from two patients identified as ataxia telangiectasia (AT), two obligate AT heterozygotes and two controls (healthy subjects with no known genetic disease or relationship to AT patients) were compared with respect to the induction of chromosomal breaks by acute and chronic -irradiation. Although there was a considerable increase in the frequency of chromosomal breaks per cell in the LCLs of AT patients resulting from acute irradiation, the small increase occurring in the LCLs of the AT heterozygotes made it difficult to distinguish them from the controls. Following chronic -irradiation, however, the frequency of chromosomal breaks per cell in the LCLs of the AT heterozygotes occupied a significantly distinct position from that of the controls. These observations suggested that the use of chronic irradiation may be a better choice in the cytogenetic characterization of AT heterozygotes.     

Effects of γ-irradiation on Na,K-ATPase in cardiac sarcolemma

Previous studies showed that adverse effect of ionizing radiation on the cardiovascular system is beside other factors mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. One of the structures highly sensitive to radicals is the Na,K-ATPase the main system responsible for extrusion of superfluous Na⁺ out of the cell which utilizes the energy derived from ATP. The aim of present study was the investigation of functional properties of cardiac Na,K-ATPase in 20-week-old male rats 6 weeks after γ-irradiation by a dose 25 Gy (IR). Irradiation induced decrease of systolic blood pressure from 133 in controls to 85 mmHg in IR group together with hypertrophy of right ventricle (RV) and hypotrophy of left ventricle (LV). When activating the cardiac Na,K-ATPase with substrate, its activity was lower in IR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (V _max) by 40 % with no changes in the value of Michaelis–Menten constant (K _m). During activation with Na⁺, we observed a decrease of the enzyme activity in hearts from IR at all tested Na⁺ concentrations. The value of V _max decreased by 38 %, and the concentration of Na⁺ that gives half maximal reaction velocity (K _Na) increased by 62 %. This impairment in the affinity of the Na⁺-binding site together with decreased number of active Na,K-ATPase molecules, as indicated by lowered V _max values, are probably responsible for the deteriorated efflux of the excessive Na⁺ from the intracellular space in hearts of irradiated rats.     

Alleviation of salt stress by low dose γ-irradiation in rice

The effects of salt stress on the growth, photosynthesis, and antioxidative ability of the rice (Oryza sativa L.) plants raising from -irradiated seeds were investigated using two cultivars, Ilpumbyeo and Sanghaehyanghyella. The 50 and 100 mM NaCl solutions caused a remarkable decrease of the early germination rate and seedling growth. However, the salt stress-induced inhibition of the growth was significantly alleviated in the -irradiated plants. The chlorophyll contents and the effective quantum yield of photosystem 2 ( _{PS 2}) were lower in the NaCl-treated plants than in the control ones, while the non-photochemical quenching was higher in the former ones. Activities of the antioxidant enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased with increasing NaCl concentrations, and the irradiated groups had even higher SOD and APX activities than the non-irradiated ones. These alleviation effects were observed similarly in both the cultivars tested.     

In vivo effects of γ-irradiation on the functional architecture of the lampbrush chromosomes in Pleurodeles (Amphibia,Urodela)

In vivo irradiation of ovaries of Pleurodeles poireti by -rays leads to structural rearrangement of lampbrush chromosomes in late vitellogenic oocytes (stages V and VI). The loops collapse into the chromomeres and the axes condense. Doses between 200 and 2,000 rads have been tested. We observed that such changes were dependent on the irradiation dose though the chronological order of the events was irrespective of the dose. The maximum effect was attained about 10 h after irradiation. The alterations are totally reversible. Over a period of 3 days, chromosomes gradually relax, regenerate loops and recover their normal appearance. In mid vitellogenic oocytes (stages III and IV) lampbrush chromosomes do not undergo radiation induced alterations. It seems that only full-grown oocytes are competent to respond to the ionizing-flow.