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PubMed2006

Cellular restriction factors affecting the early stages of HIV replication.

Several innate immune mechanisms exist in mammalian cells that prevent the replication of viruses. These cellular factors influence the tropism of retroviruses in mammalian cells by inducing a dominant restriction that acts after viral entry but before integration into the host genome. The identification of several cellular factors involved with the post entry block of HIV has recently been revealed. These recent advances identified the tripartite motif protein 5alpha (Trim5alpha) and the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G), which work to inactivate several retroviruses including HIV-1. The mechanism of restriction by these cellular proteins is unknown. Therefore, this review highlights recent advances in understanding the function of Trim5alpha and APOBEC3G.

APOBEC-1 Deaminase↗
PubMed1987

Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells.

We have critically evaluated various modifications of the alkaline elution methodology that were required to adapt the method for measuring DNA damage in cells from animal tissues treated in vivo. These modifications involved the use of a fluorometric assay for the eluted DNA using the dye Hoechst 33258, which in turn required the use of a different combination of filter and lysis conditions than those used in conventional assays. This protocol was compared with the conventional protocols by examining the DNA damage produced in cultured Chinese hamster ovary cells after treatment with three agents (gamma-rays, cis-dichlorodiammineplatinum (DDP) and trans-DDP) that differ widely in the type and repairability of the DNA lesions that they induce. For both gamma-rays and trans-DDP, the results obtained by the various protocols were equivalent with respect to the amount, type, and rate of repair of the DNA damage produced. On the other hand, for cis-DDP, where the repair time for DNA crosslinks was significantly long relative to the cell-cycle time, DNA replication appeared to be a potentially complicating factor in the measurement of crosslink repair. However, even after treatment of rapidly dividing cultured cells, where any discrepancy between the radioactivity and Hoechst assays due to DNA replication should be maximal, the resulting difference in the amount of repair measured using the two assays was relatively small. Finally, in experiments using cis-DDP and trans-DDP, the data suggested that when polycarbonate and polyvinyl chloride filters were compared using the same cell lysis conditions, their relative sensitivity to detect DNA-protein versus DNA-interstrand crosslinking were comparable. The modified alkaline elution protocol for the measurement of DNA damage in vivo therefore appears, in most cases, to produce results comparable with those obtained by the conventional protocols.

Animals↗
PubMed1990

A microplate version of the DNA-synthesis inhibition test for rapid detection of DNA-alteration potentials.

A microplate version of the DNA-synthesis inhibition test (DIT) for fast detection of DNA-alteration potentials has been developed. The DIT is based on the concept that DNA damage causes inhibition of DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents. In this test procedure human tissue culture cells (HeLa S3), prelabeled with [14C]thymidine, arfe exposed for 90 min to the substances in question. After the cells are rinsed, they are allowed to recover for 2 1/2 h in fresh culture medium, thereby unspecific interactions interfering with DNA replication are practically eliminated. Next, [3H]thymidine is added for 30 min, and then the cells are harvested and thoroughly rinsed. Finally, incorporated radioactivity is determined by liquid scintillation counting for measurement of the 3H/14C ratio. This allows for the evaluation of DNA synthesis during the 3H-labeling period and of the extent of genotoxic damage. This microplate version of the DIT can be carried out fully automated in a laboratory workstation. The test is compared to other tests for genotoxicity. Its advantages are discussed.

Autoanalysis↗
PubMed1985

Adrenal dysfunction in glycerol kinase deficiency.

The infantile form of glycerol kinase deficiency appears to be an X-linked disorder which is consistently characterized by developmental delay and adrenal cortical insufficiency and hypoplasia. We propose that the inherited deficiency of outer mitochondrial membrane-bound glycerol kinase restricts glycerophospholipid synthesis, and, hence, the activation of steroidogenesis. This would limit the conversion of cholesterol to pregnenolone, the precursor for glucocorticoids in the adrenal cortex. The deficiency in cortisol production, with a lack of feedback to the pituitary, would result in increased ACTH production and hypertrophy of the fascicular zone at the same time that replication of the cells within this zone would be inhibited. Similarly, the decreased mineralocorticoid production by the sparse glomerulosal zone would limit the ability of the individual to respond to stress, and would result in development of potentially fatal hyponatremia and hyperkalemia. Organization of the pathway for glycerophospholipid synthesis at the outer mitochondrial membrane would make this pathway particularly vulnerable to mutations disrupting the compartmented production of the parent compound, glycerol 3-phosphate, by mitochondrial-bound glycerol kinase.

Adrenal Cortex↗
PubMed1991

Spatial memory processing during hippocampal long-term potentiation in rats.

It was investigated in rats whether hippocampal long-term potentiation (LTP) influences working and/or reference memory processing in the radial maze. After preliminary training to an intermediate level of performance, experimental subjects received a series of high-frequency trains of electrical pulses applied to the right perforant path. Two control groups were adopted in order to control for possible effects of stimulation plus operation, and operation alone, respectively. Twenty-four hours after the experimental treatment, animals were administered one trial of radial maze training. This sequence of hippocampal stimulation and radial maze training was replicated 15 times. After a retention interval of 2 months, one radial maze trial was presented on each of 3 consecutive days. The analysis of field potential data showed that periodic LTP stimulation produced a state of hippocampal LTP confined to the initial portion of the acquisition phase. Evaluation of radial maze data revealed a marginal improvement of working memory performance in the experimental group during the rising phase of hippocampal LTP.

Animals↗
PubMed1985

DNA synthesis in the heterokaryons of non-dividing differentiated cells and culture cells with various proliferative potentials.

Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1/2 cells ('immortal' cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1/2, NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.

Animals↗
PubMed1977

Failure of exogenous LH to prevent PGF2alpha-induced luteolysis in beef cows.

Henderson and McNatty (Prostaglandins 9:779, 1975) proposed that LH from the preovulatory LH surge attached to receptors on luteal cells and that this attachment might protect the early corpus luteum from PGF2alpha induced luteolysis. To test this hypothesis, experiments were performed on heifers at day 10-12 of the cycle. Both jugular veins were catheterized and infusions of either saline (0.64 ml/min) or LH-NIH-B9 (10 microgram/min; 0.64 ml/min) were given. Saline infusions were from 0-12 h; LH infusions were for 10 h and were preceded by a 2 h saline infusion. All animals were given 25 mg PGF2alpha im at 6 h (6 h into the saline infusion and 4 h into the LH infusion). Blood samples were taken at 0.5 h, 1 h and 4 h intervals from 0-12h, 13-18 h and 12-42 h respectively. Serum was assayed for LH and progesterone by radioimmunoassay methods. Two animals received saline and two received LH in each experiment. Each treatment was replicated 6 times. LH infusion resulted in a mean serum LH of 75 ng/ml compared to 0.90 ng/ml in saline infused animals. This elevation of LH did not alter PGF2alpha induced luteolysis as indicated by decline in serum progesterone. This experiment does not support the hypothesis that the newly formed corpus luteum is resistant to PGF2alpha because of protection afforded by the proestrus LH surge.

Animals↗
PubMed1989

Centromere structure and function in neoplasia.

The mammalian centromere plays an essential role in maintenance of diploidy in the cell. It is therefore imperative that we understand the structure and function of the mammalian centromere in order to plan strategy to control the incidence of aneuploidy and resultant malformations of the nonneoplastic as well as neoplastic tissues. Even though considerable information is available about the structure and some functional aspects of centromeres of lower eukaryotes such as yeast, the structure of the mammalian centromere is still a matter of conjecture limited to an understanding of the base composition of the alphoid sequences putatively located in the centromeric DNA of higher apes. We do, however, have a better understanding of the structure and role of the kinetochore. In all eukaryotes analyzed so far, the centromeres in a given genome separate in a sequential manner dependent upon the time of replication of pericentric and centromeric DNA. Some chromosomes, generally found in neoplastic cells, that carry more than one centromere show premature separation of the accessory centromeres. These centromeres and the associated pericentric regions replicate their DNA in an earlier part of the S phase than those that show kinetochore activity; both, however, carry DNA of the same composition. The active centromeres in these chromosomes show kinetochore protein binding as detected by antikinetochore antibody; the inactive centromeres are usually devoid of these proteins. The double minutes in neoplastic cells also lack kinetochore proteins, perhaps due to a lack of any centromere. Some dicentric and multicentric chromosomes in cancer cells and transformed cell lines do not display premature centromere separation. In these chromosomes, all centromeric sites show kinetochore proteins and all centromeric regions replicate their DNA simultaneously. These chromosomes also exhibited meiotic-like behavior of some centromeres and show postanaphase separation of some centromeres, resulting in bridges. These bridges, upon breakage and rejoining of sister chromatids, generate new multicentric chromosomes. The resulting chromosomes also exhibit formation of compound kinetochores. Some of these phenomena are novel descriptions of the centromere behavior in cancer cells. This review also discusses the role of aberrant centromere separation in human biology, providing correlates between errors of centromere separation and neoplasia.

Animals↗
PubMed1993

Sequence of centromere separation. Minor satellite DNA does not influence separation of inactive centromeres in transformed cells of mouse.

Neoplastic cells may carry inactive centromeres on some multicentric, yet stable, chromosomes. We report that some inactive centromeres in L929 mouse cells do not contain minor satellite DNA, the DNA fraction which has been suggested to constitute the centromere. We compared the sequence of separation of inactive centromeres carrying the minor satellite with those lacking this fraction. The sequence of separation appears to be independent of whether or not the inactive centromeres carry the minor satellite DNA. The timing of replication of the inactive centromeres is also independent of this DNA. Hence, minor satellite of mouse is not a factor in holding together the subunits of inactive centromeres. Extension of these results to active centromeres might suggest that the minor satellite DNA is not a factor responsible for adhesion of the two centromere sub-units up until late meta-anaphase.

Animals↗
PubMed1992

X-chromosome inactivation and cell memory.

Mammalian X-chromosome inactivation is an excellent example of the faithful maintenance of a determined chromosomal state. As such, it may provide insight into the mechanisms for cell memory, defined as the faithful maintenance of a determined state in clonally derived progeny cells. We review here the aspects of X-chromosome inactivation that are relevant to cell memory and discuss the various molecular mechanisms that have been proposed to explain its occurrence, with emphasis on DNA methylation and a recently proposed mechanism that depends on the timing of replication.

Animals↗
PubMed1996

DNA replication in the malaria parasite.

Malaria is increasing as a global problem. Many of the drugs that were effective earlier in this century are now becoming obsolete as the parasite develops resistance to them and, despite earlier hopes, an affordable and effective vaccine remains elusive. It is hoped that a deeper understanding of the parasite's cell and molecular biology will give us a resource for the future and help us to achieve effective control. One aspect of parasite metabolism that has been the subject of recent studies is DNA replication: its timing during parasite development, the enzymes involved and the genes encoding them. In this review John White and Brian Kilbey report on the present status of these studies.

Journal Article↗
PubMed1987

Effects of therapeutically induced affect arousal on depressive symptoms, pain and beta-endorphins among rheumatoid arthritis patients.

The relationship among therapeutically induced affective arousal, depressive symptoms, pain and beta-endorphin levels were explored on 6 patients with chronic, active rheumatoid arthritis. An ABA, n of 1 study methodology was utilized, replicated 5 times. This procedure allowed the analysis of individualized changes across time in response to the therapeutic regimen. The results indicated that the treatment regimen activated the beta-endorphin system, particularly during the early and late phases of treatment. However, beta-endorphin response had little effect on reports of subjective pain. Depressive symptoms were affected positively by the treatment but were not strongly correlated to the beta-endorphin response. The results suggest that pain and depression represent independent systems and that beta-endorphin levels serve more as stress markers than analgesics in chronic, organic pain.

Adult↗
PubMed1995

Dietary protein for young grazing sheep: interactions with gastrointestinal parasitism.

Effects of protein supplementation and of nematode control on production responses in young grazing sheep and on nematode population dynamics were assessed. Young Merino wether sheep (n = 270) were allocated to one of three supplementation (Su) treatments and one of three regimes of nematode control (drench, Dr) in a factorial design. Each of the nine treatments was replicated three times in a randomised complete block. Animals received no supplement (Su0) or were given supplements at a level equivalent to 200 g day-1, fed three times a week. The supplements contained 25% lucerne meal with either 75% sunflower meal (Su1) or 75% formaldehyde-treated sunflower meal (Su2). Nematode control regimes were: not drenched unless survival of individual animals was threatened (Dr0); drenched according to a strategic drenching programme (Dr1); treatment with a controlled-release albendazole capsule (Dr2). Infections with Haemonchus contortus were suppressed by use of closantel. Due to drought conditions and lack of pasture growth, lucerne hay was fed to all sheep from Week 14 onwards (350 g per head per day, fed twice a week). Supplementation reduced the need for 'survival drenching' in Dr0 sheep considerably. Live-weight gain was increased significantly by supplementation with Su1 or Su2 during the 36-week experiment. Undrenched animals given Su1 or Su2 tended to grow faster than unsupplemented animals in the Dr1 and Dr2 groups. Greasy wool production and fibre diameter were increased by supplementation and anthelmintic treatment. Faecal worm egg counts (FEC) in undrenched sheep were significantly lower for Su1 and Su2 sheep than for Su0 sheep. There were no effects of supplementation on FEC in Dr1 or Dr2 sheep. Worm burden was generally unaffected by supplementation, while the drenching treatments Dr1 and Dr2 reduced worm burden at Week 16 and to a lesser extent at Week 27. Trichostrongylus colubriformis was the predominant nematode species, with smaller numbers of Nematodirus spp., Trichostrongylus vitrinus, Trichostrongylus axei and Ostertagia circumcincta also present. It was concluded that supplementary feeding with protein meal substantially reduced production losses attributable to nematode infections in young grazing sheep. This appears to be due to an enhanced resilience of the host, rather than any major changes in development of protective immunity.

Animal Feed↗
PubMed2005

Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection.

The objective of this study was to compare the effectiveness of different methods of bovine oocyte activation following intracytoplasmic sperm injection (ICSI) in terms of oocyte cleavage and blastocyst rates, and calf production. Oocytes were harvested, post mortem, from the ovaries of Japanese Black heifers or cows. ICSI was carried out using a piezo-electric actuator. The injected or sham-injected oocytes that were assigned to three activation treatments, each replicated three times, were studied: (1) exposure to 5 microM ionomycin for 5 min (ionomycin); (2) exposure to 5 microM ionomycin for 5 min followed by culture in TCM199 for 3 h and a further 3h culture in 1.9 mM 6-dimethylaminopurine (DMAP-ionomycin+DMAP); (3) exposure to 7% ethanol in TCM199 for 5 min, 4 h after ICSI (ethanol). One or two blastocysts from the ionomycin+DMAP (8 recipients) and ethanol (17 recipients) oocyte activation treatments were non-surgically transferred into Holsteins for the study of calf production. The highest cleavage and blastocyst production rates were observed in the ionomycin+DMAP treatment (83.9% and 40.1%) by the ICSI. These rates were significantly (P<0.05) higher than those for the ionomycin oocyte activation treatment (57.6% and 18.2%) but did not differ from the ethanol treatment (75.6% and 29.4%). In the sham-injected, the highest blastocyst production rates were observed for the ionomycin+DMAP and ethanol treatments (10.7% and 11.3%). Pregnancy and birth rates for blastocysts derived from the ethanol oocyte activation treatment (58.8% and 47.4%) were significantly higher (P<0.05) than those of the ionomycin+DMAP treatment (12.5% and 9.2%). The results showed that post-ICSI oocyte activation with ethanol is more effective than activation with ionomycin alone or with ionomycin+DMAP for the production of viable blastocysts and calves.

Adenine↗
PubMed2004

Influences of vermicomposts on field strawberries: 1. effects on growth and yields.

Vermicomposts processed commercially from food wastes and paper wastes were applied, to 4.5 m(2) field plots, under high plastic hoop tunnels, at rates of 5 or 10 tha(-1) to evaluate their effects on the growth and yields of strawberries (Fragaria ananasa) var. 'Chandler'. The vermicomposts were incorporated into the top 10 cm of soil and supplemented, based on chemical analyses, with amounts of inorganic NPK fertilizers calculated to equalize the initial fertilizer rates of 85-155-125 kgha(-1) NPK applied to the inorganic fertilizer plots. All treatments were replicated four times, in a completely randomized design, at two field sites on Doles silt loam or Hoytville silty clay loam at Piketon and Fremont, Ohio, respectively. Vermicompost applications increased strawberry growth and yields significantly; including increases of up to 37% in leaf areas, 37% in plant shoot biomass, 40% in numbers of flowers, 36% in numbers of plant runners and 35% in marketable fruit weights. These responses seemed not to be dose-dependent, since strawberries at one site grew fastest and yielded most in response to the 10 tha(-1) vermicompost application rate, whereas they responded positively and similarly to both the 5 and 10 tha(-1) rates of applications at the other site. These responses could not have been mediated by availability of macronutrients, since all plots were supplemented with inorganic fertilizers, to equalize macronutrient inputs for all treatments, but based on other research in our laboratory could have been due to production of plant growth regulators by microorganisms during vermicomposting.

Agriculture↗
PubMed2006

The influence of manure composition on emissions of odour and ammonia from finishing pigs fed different concentrations of dietary crude protein.

An investigation was conducted into the influence of manure composition on the odour emission rate (OER) and the emission rate of ammonia (NH(3)), when diets containing 130, 160, 190 and 210gkg(-1) crude protein (CP) were fed to finishing pigs. A group of four boars and four gilts, housed in environmentally sealed pens, were assigned to each diet for a 23-day experimental period which was replicated three times (n=3). Ventilation air from each pen was sampled for NH(3) and odour, by olfactometry, on four days during the trial period. Simultaneous collections of manure were taken from the surface and base of each pit. The pH and the concentrations of dry matter, total Kjeldahl nitrogen (TKN), total ammoniacal nitrogen (TAN) and volatile fatty acids in the manure were measured. Manure composition differed between samples from the surface and base of the pit (P<0.05). Reducing dietary CP concentration decreased the emission of NH(3) (linear, P<0.001). The acetic acid:propionic acid ratio in manure samples was correlated to OER (r=0.79, P<0.001). There was a quadratic relationship between dietary CP concentration and OER (P<0.05). OER decreased between 210gkg(-1) and 160gkg(-1) CP and increased between 160gkg(-1) and 130gkg(-1) CP. In conclusion, reducing dietary crude protein levels could be used effectively to reduce ammonia emissions and OER, although no significant advantage was to be gained in OER from reducing crude protein level below 160gkg(-1).

Ammonia↗