Ovine TRIM5α Can Restrict Visna/Maedi Virus

The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.     

Serotonin uptake in the central nervous system of rats fed a corn-diet

1. Endogenous serotonin (5-HT), 5-hydroxyindol acetic acid (5-HIAA) content and exogenous 5-HT uptake (Km and Vmax) were measured in different brain regions (cerebellum, diencephalon, brain stem and telencephalon) of rats fed with a corn diet and restricted protein (8%) diet during 6 weeks. 2. A reduction of 5-HT levels was found in all regions studied of animals fed a corn diet, whereas, 5-HIAA was only decreased in brain stem and diencephalon. 3. An important increase in Km and Vmax were registered in brain stem and diencephalon of protein restricted animals, whereas, an increase of 5-HT uptake affinity in cerebellum, brain stem and telencephalon (35, 42 and 33% respectively) was observed. Simultaneously, under corn diet conditions, the Vmax decreased 40, 30 and 34% respectively in those regions. 4. It is suggested that the brain stem was the more sensitive area under nutritional restricted conditions and the development of some possible compensatory mechanisms of the 5-HTergic system is discussed.     

Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors

Radial glia are among the first cells that develop in the embryonic central nervous system. They are progenitors of glia and neurons but their relationship with restricted precursors that are also derived from neuroepithelia is unclear. To clarify this issue, we analyzed expression of cell type specific markers (BLBP for radial glia, 5A5/E-NCAM for neuronal precursors and A2B5 for glial precursors) on cortical radial glia in vivo and their progeny in vitro. Clones of cortical cells initially expressing only BLBP gave rise to cells that were A2B5+ and eventually lost BLBP expression in vitro. BLBP is expressed in the rat neuroepithelium as early as E12.5 when there is little or no staining for A2B5 and 5A5. In E13.5-15.5 forebrain, A2B5 is spatially restricted co-localizing with a subset of the BLBP+ radial glia. Analysis of cells isolated acutely from embryonic cortices confirmed that BLBP expression could appear without, or together with, A2B5 or 5A5. The numbers of BLBP+/5A5+ cells decreased during neurogenesis while the numbers of BLBP+/A2B5+ cells remained high through the beginning of gliogenesis. The combined results demonstrate that spatially restricted subpopulations of radial glia along the dorsal-ventral axis acquire different markers for neuronal or glial precursors during CNS development.     

A HLA-DQ5 restricted Melan-A/MART-1 epitope presented by melanoma tumor cells to CD4+ T lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen expressed by tumor cells of the majority of melanoma patients and, as such, is considered as a good target for melanoma immunotherapy. Nonetheless, the number of class I and II restricted Melan-A epitopes identified so far remains limited. Here we describe a new Melan-A/MART-1 epitope recognized in the context of HLA-DQa1*0101 and HLA-DQb1*0501, -DQb1*0502 or -DQb1*0504 molecules by a CD4+ T cell clone. This clone was obtained by in vitro stimulation of PBMC from a healthy donor by the Melan-A51-73 peptide previously reported to contain a HLA-DR4 epitope. The Melan-A51-73 peptide, therefore contains both HLA-DR4 and HLA-DQ5 restricted epitope. We further show that Melan-A51-63 is the minimal peptide optimally recognized by the HLA-DQ5 restricted CD4+ clone. Importantly, this clone specifically recognizes and kills tumor cell lines expressing Melan-A and either HLA-DQb1*0501, -DQb1*0504 or -DQb1*0502 molecules. Moreover, we could detect CD4+ T cells secreting IFN-gamma in response to Melan-A51-63 and Melan-A51-73 peptides among tumor infiltrating and blood lymphocytes from HLA-DQ5+ patients. This suggests that spontaneous CD4+ T cell responses against this HLA-DQ5 epitope occur in vivo. Together these data significantly increase the fraction of melanoma patients susceptible to benefit from a Melan-A class II restricted vaccine approach.     

Synthesis and biological evaluation of conformationally restricted and nucleobase-modified analogs of the anticancer compound 3'-C-ethynylcytidine (ECyd)

A series of conformationally restricted and nucleobase-modified analogs of the anticancer compound 3'-C-ethynylcytidine (ECyd) and its uracil analog (EUrd) have been synthesized. While none of the conformationally restricted analogs displayed anticancer activity, 5-iodo-EUrd and 5-bromo-EUrd displayed potent anticancer activity with IC50 values of 35 nM and 0. 73 microM.     

SYNTHESIS AND BIOLOGICAL EVALUATION OF CONFORMATIONALLY RESTRICTED AND NUCLEOBASE-MODIFIED ANALOGS OF THE ANTICANCER COMPOUND 3′-C-ETHYNYLCYTIDINE (ECYD)

A series of conformationally restricted and nucleobase-modified analogs of the anticancer compound 3′-C-ethynylcytidine (ECyd) and its uracil analog (EUrd) have been synthesized. While none of, the conformationally restricted analogs displayed anticancer activity, 5-iodo-EUrd and 5-bromo-EUrd displayed potent anticancer activity with IC50 values of 35 nM and 0.73 μM.     

Growth performance and body composition in response to dietary protein and lipid levels in Nile tilapia (Oreochromis niloticus Linnaeus, 1758) subjected to normal and temporally restricted feeding regimes

A factorial experiment was designed to examine the effect on compensatory growth (CG) of Nile tilapia Oreochromis niloticus fed diets containing different protein and lipid levels under normal and temporally restricted feeding regimes. Four diets were formulated to contain either 30% or 36% crude protein, and 5% or 11% crude lipid. Triplicate replicates of each treatment were assigned to 24 150‐L tanks (20 fish/tank density). Fish (mean initial weight ± SD = 8.79 ± 0.34 g) were then fed either the normal feeding regime (thrice daily to apparent satiation) or the restricted regime (1 day feed deprivation followed by 3 days of feeding to apparent satiation) over a 44‐days study period. Fish receiving a diet under the restricted regime achieved weight gains (WG) comparable to fish consuming the diet containing 30% protein and 5% lipids under the normal feeding regime. Fish maintained on the restricted feeding regime exhibited reduced feed intake (FI), WG, feed efficiency ratio (FE), protein efficiency rate (PER) and hepatosomatic index versus fish on the normal feeding regime, except WG in fish fed the diet with 30% protein and 5% lipids. However, the resultant FI (85%~94%) was higher than the excepted 75% intake when fish were subjected to the restricted regime. Feeding 11% lipid diets led to improved FI, WG, FE, and PER compared to feeding the 5% lipid diets. Increased FI, WG, and FE, but reduced PER were observed in fish fed with 36% protein versus fish fed 30% protein. Fish receiving the 36% protein diets had lower whole‐body moisture and ash contents, but elevated whole‐body protein and lipid contents compared to those receiving the 30% protein diets. Whole‐body moisture contents were lower, but whole‐body protein, lipid and ash contents were higher in fish fed 11% lipid diets than in fish fed 5% lipid diets. There was an increase in whole‐body moisture content, but a decrease in protein and lipid content in response to the restricted feeding regime. Ash content was not affected by the feeding regime. The present study shows that Nile tilapia fed diets subjected to a restricted feeding regime exhibited growth comparable to those fed the diet at 30% protein and 5% lipid levels under a normal feeding regime. This positive effect was more pronounced in diets at a high protein level or in a combination of high protein and lipid levels.     

Higher order structure of the ribosomal 5 S RNA.

The structure of the ribosomal 5 S RNA was examined using Fe(II)-EDTA, a solvent-based reagent that cleaves the phosphodiester backbone of both double- and single-stranded RNA but is restricted by the three-dimensional structure. In the yeast 5 S RNA, cleavages were significantly restricted in six specific regions of the molecule; restrictions in only two of these regions were clearly dependent on a high salt/magnesium ion environment. A comparison of four RNAs of diverse origin revealed strong similarities in the cleavage profiles supporting a highly conserved higher order structure. Taken together with previous studies these data provide a more detailed modeling of the three-dimensional structure.     

Restriction of foamy viruses by primate Trim5alpha

Foamy viruses (FVs) are unconventional retroviruses with a replication strategy that is significantly different from orthoretroviruses and bears some homology to that of hepadnaviruses. Although some cellular proteins, such as APOBEC3, have been reported to block FVs, no restriction by Trim5alpha has been described to date. The sensitivity of three FV isolates of human-chimpanzee or prototypic (PFV), macaque (SFVmac), and feline (FFV) origin to a variety of primate Trim5alphas was therefore tested. PFV and SFVmac were restricted by Trim5alphas from most New World monkeys, but not from other primates, whereas FFV-based vectors were restricted by Trim5alphas from the great apes gorilla and orangutan. Trim5alphas from Old World monkeys did not restrict any FV isolate tested. Capuchin Trim5alpha was unique, as it restricted SFVmac and FFV but not PFV. Trim5alpha specificity for FVs was determined by the B30.2 domain, interestingly involving, in some instances, the same residues of the variable regions previously implicated as major determinants for human immunodeficiency virus type 1 restriction. FVs with chimeric Gags were made to map the viral determinants of sensitivity to restriction. The N-terminal half of the Gag molecule was found to contain the regions that control susceptibility. This region most likely corresponds to the capsid of conventional retroviruses. Due to their unique replication strategy, FVs should provide a valuable new system to examine the mechanism of retroviral restriction by Trim5alpha.     

Feeding and growth of whitefish fed restricted and abundant rations: influences on growth heterogeneity and brain serotonergic activity

The whitefish, Coregonus lavaretus , is a schooling species, and it was hypothesized that, when faced with feed restriction, the behaviours associated with a schooling habit would lead to there being less heterogeneity in feeding and growth than has been observed in other members of the Salmonidae. This was tested by monitoring the feeding, growth and brain serotonergic activity of whitefish subjected to different levels of feed restriction (full rations and regimes estimated to give a 25 and 50% reduction in growth rate). The feed restrictions were successful in reducing rates of feeding and growth, but these reductions were not accompanied by any marked increases in interindividual variations in either feed intake or growth rates. Consequently, the imposition of restricted feeding regimes did not result in an increase in the size variation of fish within rearing tanks. Further, the brain serotonergic activity (assessed as 5-HIAA: 5-HT ratio) of individual fish was not found to correlate with either their feed intake or growth rates. This suggests that any competition resulting from restricted feeding did not lead to the establishment of marked dominance-subordination relationships. 5-HIAA: 5-HT ratios were, however, higher amongst fish fed the most restricted rations than in fish of the other groups. This may have been a reflection of a general nutritional stress rather than pointing to an increase in social conflict amongst the fish fed the lowest rations. When provided with abundant rations the fish that had been feed-restricted responded by becoming hyperphagic and showing catch-up growth, the degree of compensation being related to the severity of the previous food restriction. Taken together the results suggest that restricted feeding may be a suitable method for controlling rates of production of whitefish, without there being a high risk of a marked increase in size variation with the passage of time.     

Enhanced Food Anticipatory Activity Associated with Enhanced Activation of Extrahypothalamic Neural Pathways in Serotonin2C Receptor Null Mutant Mice

The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin_2C receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enhanced food anticipatory activity compared to wild-type littermates, without phenotypic differences in the impact of restricted feeding on food consumption, body weight loss, or blood glucose levels. Moreover, we show that the enhanced food anticipatory activity in 5-HT2CR null mutant mice develops independent of external light cues and persists during two days of total food deprivation, indicating that food anticipatory activity in 5-HT2CR null mutant mice reflects the locomotor output of a food-entrainable oscillator. Whereas restricted feeding induces c-fos expression to a similar extent in hypothalamic nuclei of wild-type and null mutant animals, it produces enhanced expression in the nucleus accumbens and other extrahypothalamic regions of null mutant mice relative to wild-type subjects. These data suggest that 5-HT2CRs gate food anticipatory activity through mechanisms involving extrahypothalamic neural pathways.     

Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems

The activities of rat intestinal enzymes, sucrase, lactase, maltase, trehalase, γ-glutamyltransferase, leucylnaphthylamide-hydrolyzing activity, and the transport system for glucose follow diurnal rhythms on ad libitum and restricted feeding regimes. In response to 6 days of restricted feeding, food available between 1400 and 1800 Eastern Standard Time, all rhythms shifted in time and the daily levels of activities were changed. Alkaline phosphatase activity followed a diurnal rhythm only in restricted fed animals.In restricted fed rats several activity patterns were observed, some with short periods of maximum activity, 3 h or less, and some with plateaus of maximum activity, 5–9 h long. In respect to the time of day of the synchronizer, sucrase peaked before feeding, glucose transport peaked during feeding, alkaline phosphatase peaked after feeding, and the other enzymes had higher levels of activity before, during and after feeding. The effect of restricted feeding on the daily activity levels were: a decrease in leucylnaphthylamide-hydrolyzing activity, no change in alkaline phosphatase, and increases in the others.These enzyme and transport systems exhibit a large amount of individual regulation or control as reflected by the lack of a uniform activity pattern and response to the synchronizer, and the variation in direction and magnitude of the adaptations to restricted feeding.     

Z-Spectrum Analysis Provides Proton Environment Data (ZAPPED): A New Two-Pool Technique for Human Gray and White Matter

A new technique – Z-spectrum Analysis Provides Proton Environment Data (ZAPPED) – was used to map cross-relaxing free and restricted protons in nine healthy subjects plus two brain tumor patients at 3T. First, MT data were acquired over a wide symmetric range of frequency offsets, and then a trio of quantitative biomarkers, i.e., the apparent spin-spin relaxation times (T_2,f, T_2,r) in both free and restricted proton pools as well as the restricted pool fraction F_r, were mapped by fitting the measured Z-spectra to a simple two-Lorentzian compartment model on a voxel-by-voxel basis. The mean restricted exchangeable proton fraction, F_r, was found to be 0.17 in gray matter (GM) and 0.28 in white matter (WM) in healthy subjects. Corresponding mean values for apparent spin-spin relaxation times were 785 µs (T_2,f) and 17.7 µs (T_2,r) in GM, 672 µs (T_2,f) and 23.4 µs (T_2,r) in WM. The percentages of F_f and F_r in GM are similar for all ages, whereas F_r shows a tendency to decrease with age in WM among healthy subjects. The patient ZAPPED images show higher contrast between tumor and normal tissues than traditional T₂-weighted and T₁-weighted images. The ZAPPED method provides a simple phenomenological approach to estimating fractions and apparent T₂ values of free and restricted MT-active protons, and it may offer clinical useful information.     

Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems.

The activities of rat intestinal enzymes, sucrase, lactase, maltase, trehalase, gamma-glutamyltransferase, leucylnaphthylamide-hydrolyzing activity, and the transport system for glucose follow diurnal rhythms on ad libitum and restricted feeding regimes. In response to 6 days of restricted feeding, food available between 1400 and 1800 Eastern Standard Time, all rhythms shifted in time and the daily levels of activities were changed. Alkaline phosphatase activity followed a diurnal rhythm only in restricted fed animals. In restricted fed rats several activity patterns were observed, some with short periods of maximum activity, 3 h or less, and some with plateaus of maximum activity, 5-9 h long. In respect to the time of day of the synchronizer, sucrase peaked before feeding, glucose transport peaked during feeding, alkaline phosphatase peaked after feeding, and the other enzymes had higher levels of activity before, during and after feeding. The effect of restricted feeding on the daily activity levels were: a decrease in leucylnaphthylamide-hydrolyzing activity, no change in alkaline phosphatase, and increases in the others. These enzyme and transport systems exhibit a large amount of individual regulation or control as reflected by the lack of a uniform activity pattern and response to the synchronizer, and the variation in direction and magnitude of the adaptations to restricted feeding.     

Boar sperm membranes antigens. I. Topography of a mobile glycoprotein of the sperm cell membrane

A monoclonal antibody, designated mAb P86/5, was generated by immunization of female Balb/c mice with a membrane vesicle fraction composed of the outer acrosomal membrane and plasma membrane (PM-OAM). As determined by fluorescence microscopy and electron microscopy P86/5 recognizes a sperm plasma membrane antigen that is restricted to the sperm head. In intact spermatozoa the P86/5-antigen is distributed over the surface of the sperm head with the exception of the rostral region. By comparing the antibody binding pattern generated at 4 degrees C and 25 degrees C, it could be shown that the P86/5-antigen is capable to diffuse freely within the cell membrane overlying the acrosome whereas its lateral mobility is restricted to the post-acrosomal region. The P86/5-antigen had a molecular weight of about 78 kDa as revealed by SDS-PAGE and western blotting. The glycoprotein nature of the P86/5-antigen was established by lectin affinity chromatography.     

Causal inference on the difference of the restricted mean lifetime between two groups

When comparing survival times between two treatment groups, it may be more appropriate to compare the restricted mean lifetime, i.e., the expectation of lifetime restricted to a time L, rather than mean lifetime in order to accommodate censoring. When the treatments are not assigned to patients randomly, as in observational studies, we also need to account for treatment imbalances in confounding factors. In this article, we propose estimators for the difference of the restricted mean lifetime between two groups that account for treatment imbalances in prognostic factors assuming a proportional hazards relationship. Large-sample properties of our estimators based on martingale theory for counting processes are also derived. Simulation studies were conducted to compare these estimators and to assess the adequacy of the large-sample approximations. Our methods are also applied to an observational database of acute coronary syndrome patients from Duke University Medical Center to estimate the treatment effect on the restricted mean lifetime over 5 years.     

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.