Protection from oxidative stress by methionine sulfoxide reductases in RPE cells

We investigated the role of methionine sulfoxide reductases (Msrs) in oxidant-stress-induced cell death in retinal pigmented epithelial (RPE) cells. In RPE cells exposed to varying doses of H(2)O(2), gene expression of MsrA and hCBS-1 (the human analog of MsrB2) increased in a dose-dependent and time-dependent manner with maximal increase with 150 microM H(2)O(2) in 24h. H(2)O(2) treatment resulted in the generation of reactive oxygen species and activation of caspase 3. Confocal microscopic and protein analysis showed an increase in MsrA expression in cytosol and mitochondria. Silencing of MsrA resulted in caspase 3 induction and accentuated cell death from H(2)O(2). Focal, strong immunoreactivity for MsrA was observed in sub-RPE macular drusen from patients with age-related macular degeneration. In summary, our data show that MsrA and hCBS-1 are up-regulated in oxidative stress to counteract injury to RPE.     

Eukaryotic translation initiation factor 4E is a cellular target for toxicity and death due to exposure to cadmium chloride

Whether translation initiation factor 4E (eIF4E), the mRNA cap binding and rate-limiting factor required for translation, is a target for cytotoxicity and cell death induced by cadmium, a human carcinogen, was investigated. Exposure of human cell lines, HCT15, PLC/PR/5, HeLa, and Chang, to cadmium chloride resulted in cytotoxicity and cell death, and this was associated with a significant decrease in eIF4E protein levels. Similarly, specific silencing of the expression of the eIF4E gene, caused by a small interfering RNA, resulted in significant cytotoxicity and cell death. On the other hand, overexpression of the eIF4E gene was protective against the cadmium-induced cytotoxicity and cell death. Further studies revealed the absence of alterations in the eIF4E mRNA level in the cadmium-treated cells despite their decreased eIF4E protein level. In addition, exposure of cells to cadmium resulted in enhanced ubiquitination of eIF4E protein while inhibitors of proteasome activity reversed the cadmium-induced decrease of eIF4E protein. Exposure of cells to cadmium, as well as the specific silencing of eIF4E gene, also resulted in decreased cellular levels of cyclin D1, a critical cell cycle and growth regulating gene, suggesting that the observed inhibition of cyclin D1 gene expression in the cadmium-treated cells is most likely due to decreased cellular level of eIF4E. Taken together, our results demonstrate that the exposure of cells to cadmium chloride resulted in cytotoxicity and cell death due to enhanced ubiquitination and consequent proteolysis of eIF4E protein, which in turn diminished cellular levels of critical genes such as cyclin D1.     

A new recessive ametapodia mutation in the chicken (Gallus domesticus)

An apparently new mutation that is associated with abnormal limb development appeared in a strain of Light Brown Leghorn chickens. Mutants are characterized by the complete absence of the tarsometatarsals, while severely hypoplastic development of the metacarpals is also present. The phenotype of the new mutant (ametapodia-2) closely resembles ametapodia-1, described in 1967, but ametapodia-2 is inherited as an autosomal recessive (AMET*A), while ametapodia-1 was associated with an incompletely dominant gene (MP*A). Only heterozygous ametapodia-1 (MP*N/MP*A) were viable and able to reproduce, while homozygous ametapodia-2 mutants do not normally survive beyond 2-4 days of age. The shankless mutation (SHL*S) also reduces development of the metatarsal and metacarpal bones and has been shown to be associated with a pericentric inversion of chromosome 2. No obvious cytologic abnormality was apparent in ametapodia-2 birds, and offspring of a cross between AMET*A carriers and shankless birds were normal, indicating that the two mutations are not alleles. Ametapodia-1 (MP*A) was found to be linked to the rose comb locus (R) by 16 crossover units. Linkage test matings between AMET*A and (R*R) showed independent segregation, strongly suggesting that the mutation occurred at a relatively distant locus and therefore is probably not allelic to MP*A.     

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from chickens in Grenada, West Indies

The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.     

Toxoplasmosis in a Hawaiian monk seal (Monachus schauinslandi)

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment with oocysts. T. gondii was identified in a Hawaiian monk seal (Monachus schauinslandi) that had visceral and cerebral lesions. Tachyzoites were found in the lymph nodes, spleen, diaphragm, heart, adrenal glands, and brain. A few tissue cysts were found in sections of the cerebrum. The diagnosis was confirmed serologically, by immunohistochemical staining with T. gondii-specific polyclonal rabbit serum, and by the detection of T. gondii DNA. The genotype was determined to be type III by restriction fragment length polymorphisms of the SAG2 gene. This is the first report of T. gondii infection in a Hawaiian monk seal.     

First biologic and genetic characterization of Toxoplasma gondii isolates from chickens from Africa (Democratic Republic of Congo, Mali, Burkina Faso, and Kenya)

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. In the present study, prevalence of T. gondii in chickens from Democratic Republic of Congo, Mali, Burkina Faso, and Kenya is reported. The prevalence of T. gondii antibodies in sera of 50 free-range chickens from Congo was 50% based on the modified agglutination test (MAT); antibody titers were 1:5 in 7, 1:10 in 7, 1:20 in 6, 1:40 in 1, and 1:160 or more in 4 chickens. Hearts, pectoral muscles, and brains of 11 chickens with titers of 1:20 or more were bioassayed individually in mice; T. gondii was isolated from 9, from the hearts of 9, brains of 3, and muscles of 3 chickens. Tissues of each of the 14 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice; T. gondii was isolated from 1 chicken with a titer of 1:10. Tissues from the remaining 25 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts, but none was seen. The results indicate that T. gondii localizes in the hearts more often than in other tissues of naturally infected chickens. Genotyping of these 10 isolates using the SAG2 locus indicated that 8 were isolates were type III, 1 was type II, and 1 was type I. Two isolates (1 type I and 1 type III) were virulent for mice. Toxoplasma gondii was isolated by mouse bioassay from a pool of brains and hearts of 5 of 48 chickens from Mali and 1 of 40 chickens from Burkina Faso; all 6 isolates were avirulent for mice. Genetically, 4 isolates were type III and 2 were type II. Sera were not available from chickens from Mali and Burkina Faso. Toxoplasma gondii antibodies (MAT 100 or more) were found in 4 of 30 chickens from Kenya, and T. gondii was isolated from the brain of 1 of 4 seropositive chickens; this strain was avirulent for mice and was type II. This is the first report on isolation and genotyping of T. gondii from any source from these 4 countries in Africa.     

Identification, sequence characterization, and analysis of expression profiles of three novel CC chemokines from domestic duck (Anas platyrhynchos)

Chemokines are low-molecular weight-chemotactic cytokines, which are involved in lymphocyte trafficking and migration of leucocytes to sites of injury, in immune surveillance and in healing process. They also play a role in pathogenesis of inflammatory diseases. Three novel CC chemokines were identified from domestic duck (Anas platyrhynchos) by screening of an enriched cDNA library constructed from mitogen-stimulated splenic mononuclear cells. Two of the clones (AB163 and AB330) had a very high nucleotide (both about 81%) and predicted amino acid level (71 and 76%, respectively) identity to the reported chicken macrophage inflammatory protein 1- (MIP-1; SCYA4) and regulated upon activation of normal T-cell expressed and secreted (RANTES; SCYA5) sequences. In phylogenetic analysis, these molecules clustered together with corresponding chemokines reported from other vertebrates. The third clone (AB187) had highest homology to chicken MIP-1 (36% amino acid identity) and showed closer relation to a number of chemokines belonging to monocyte chemoattractant proteins and MIP-1 chemokines. Expression of these molecules was upregulated upon mitogen stimulation of splenocytes as detected by semiquantitative RT-PCR. AB187 showed several fold increases (about 8.5 times) in the mRNA expression. Basal level expression of some of these chemokines was detected in both lymphoid and nonlymphoid tissues, including spleen, liver, lung, and bone marrow. Considering the importance of this animal species as a model for diseases such as chronic human hepatitis B, further studies will offer valuable insights into the role of these molecules in immunopathology of such diseases.The nucleotide sequences that are reported in this paper have been submitted to the NCBI Genbank database. Accession nos. AB163 (AY641435), AB187 (AY641436), and AB330 (AY641437).     

In vitro plant regeneration and genetic transformation of Dichanthium annulatum

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.     

Synthesis and in vitro pharmacological studies of new C(2) modified salvinorin A analogues

Salvinorin A is the most potent naturally occurring opioid agonist yet discovered with high selectivity and affinity for kappa-opioid receptor. To explore its structure and activity relationships, a series of salvinorin A derivatives modified at the C2 position were prepared and studied. These salvinorin A derivatives were screened for binding and functional activities at the human kappa-opioid receptor. Compound 4, containing a methoxymethyl group at the 2-position, was a full kappa-agonist with an EC50 value at 0.6 nM, which is about 7 times more potent than salvinorin A.