The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Fluorescence resonance energy transfer studies on the proximity between lysine-107 and cysteine-239 in rabbit muscle aldolase

Spatial relationships between Lys-107, which binds the C-6 phosphate group of the substrate, and fast-reacting Cys-239, located outside the active site of rabbit muscle aldolase, were studied by means of resonance energy transfer. The Lys-107 residue was covalently linked to pyridoxal phosphate (fluorescence donor) and the Cys-239 residue was modified by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (fluorescence acceptor). The energy transfer between donor and acceptor has been demonstrated. The steady-state and the lifetime measurements indicate that in solution the distance between Lys-107 and Cys-239 in the aldolase molecule is 12.4 A assuming chi 2 = 2/3.     

Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity

Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-gamma and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.     

The reef building coral Stylophora pistillata uses stored carbohydrates to maintain ATP levels under thermal stress

Coral Reefs - Coral reefs are on the brink of collapse from global warming and associated coral bleaching. Coral bleaching is the loss of algal symbionts from the coral tissue. The reduction in...     

Enhanced Responses to Tumor Immunization Following Total Body Irradiation Are Time-Dependent

The development of successful cancer vaccines is contingent on the ability to induce effective and persistent anti-tumor immunity against self-antigens that do not typically elicit immune responses. In this study, we examine the effects of a non-myeloablative dose of total body irradiation on the ability of tumor-naïve mice to respond to DNA vaccines against melanoma. We demonstrate that irradiation followed by lymphocyte infusion results in a dramatic increase in responsiveness to tumor vaccination, with augmentation of T cell responses to tumor antigens and tumor eradication. In irradiated mice, infused CD8⁺ T cells expand in an environment that is relatively depleted in regulatory T cells, and this correlates with improved CD8⁺ T cell functionality. We also observe an increase in the frequency of dendritic cells displaying an activated phenotype within lymphoid organs in the first 24 hours after irradiation. Intriguingly, both the relative decrease in regulatory T cells and increase in activated dendritic cells correspond with a brief window of augmented responsiveness to immunization. After this 24 hour window, the numbers of dendritic cells decline, as does the ability of mice to respond to immunizations. When immunizations are initiated within the period of augmented dendritic cell activation, mice develop anti-tumor responses that show increased durability as well as magnitude, and this approach leads to improved survival in experiments with mice bearing established tumors as well as in a spontaneous melanoma model. We conclude that irradiation can produce potent immune adjuvant effects independent of its ability to induce tumor ablation, and that the timing of immunization and lymphocyte infusion in the irradiated host are crucial for generating optimal anti-tumor immunity. Clinical strategies using these approaches must therefore optimize such parameters, as the correct timing of infusion and vaccination may mean the difference between an ineffective treatment and successful tumor eradication.     

Synthesis and antioxidant activity evaluation of novel antiparkinsonian agents,aminoadamantane derivatives of nitroxyl free radical

Two new analogues of the antiparkinsonian drug 1-aminoadamantane: 4-(1-adamantylamino)-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-(1-adamantylammonio)-1-hydroxy-2,2,6,6-tetramethylpiperidinium dihydrochloride have been synthesized. Their antioxidant activity towards reactive oxygen species (ROS: (z.rad;)OH and O(2)(z.rad;-)) have been evaluated in three test systems. The compound with nitroxide substituent displays higher anti-oxidative capacity than those containing hydroxylamine. The in vivo study of ROS-involving 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rat model of induced parkinsonism was undertaken to ascertain the neuroprotective ability of the novel synthesized compounds-antioxidants. The data clearly shows that the nitroxide free radical moiety of the molecule is necessary for their neuroprotective action on dopaminergic neurons under the applied conditions of deep oxidative stress caused by the neurotoxin (MPTP). The new synthesized analogues may find application in treatment of parkinsonian syndromes, either to block or to reduce the ROS-mediated neuronal damage and death.     

AGE DETERMINATION IN MANATEES USING GROWTH-LAYER-GROUP COUNTS IN BONE

Growth layers were observed in histological preparations of bones of known-age, known minimum-age, and tetracycline-marked free-ranging and captive Florida manatees ( Trichechus manatus latirostris ), substantiating earlier preliminary findings of other studies. Detailed analysis of 17 new case histories showed that growth-layer group (GLG) counts in the periotic bone were consistent with known age, or time since tetracycline administration, but were less reliable in other bones. GLG counts were also made in periotic bones of 1,196 Florida manatees of unknown age found dead from 1974 through 1991. These counts were conducted in order to assess variability and to determine relationships among estimated age, size, sex, and degree of bone resorption. Resorption can interfere with accuracy of GLG counts. This effect does not occur until ages greater than about 15 yr and body lengths greater than 300 cm are attained. GLGs were also observed in periotic bones of Antillean manatees ( Trichechus manatus manatus ) but were not validated against known-age specimens. Use of GLG counts in the periotic bone is suitable for application to studies of population dynamics and other age-related aspects of manatee biology.     

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.