Mismatch repair of deaminated 5-methyl-cytosine

Deamination of 5-methyl-cytosine in double-stranded DNA produces a G-T mismatch. Heteroduplexes of bacteriophage lambda DNA containing a G-T mismatch at the site of a G-5-meC base-pair in one of the parental phages were constructed and used to transfect Escherichia coli cells. Genetic analysis of the progeny phages derived from such heteroduplexes suggests that, in E. coli, mismatches resulting from the deamination of 5-methyl-cytosine are repaired by a system requiring the E. coli dcm methylase and some, but not all, of the functions of the E. coli methyl-directed mismatch repair system. The repair appears to act only on the G-T mismatch and acts specifically to restore the cytosine methylation sequence.     

Variability in reproductive traits in Jatropha curcas L. accessions during early developmental stages under warm subtropical conditions

Variability in floral, fruit, and seed characteristics, and oil content of 15 accession of Jatropha curcas during early development were assessed during two flowering periods in south Florida subtropical climate. The two flowering periods had leaf flushing in March. Field evaluation using 18 quantitative traits showed significant variation among accessions. The number of female flowers and female : male flower ratio ranged from 1 to 15 and 1 : 8.8 to 1 : 67.8, respectively. Fruit set by natural pollination was 89 and 66% during the first (1st) and second (2nd) flowering periods, respectively. A higher number of female‐type inflorescences were observed during summer. There were significant differences in seed traits, except for number of seeds per fruit. Accession TREC 31 had the highest individual seed dry weight and 100‐seed weight (0.83 g and 79.7 g, respectively). The oil content varied from 19.30% to 35.62%. Seed dry weight had positive correlation with seed fresh weight, seed length, seed thickness, seed width, and 100‐seed weight, but negative correlation with oil content. Based on the cluster analysis using 15 morphological traits, jatropha accessions were grouped into five main clusters and accessions from different geographic regions grouped together in a cluster. Principal component analyses (PCA) revealed morphological variation. The first three components explained 73.5% of the total variation and seed dry weight, 100‐seed weight, total flowers per inflorescence, male flowers per inflorescence and fruit set can be used to distinguish accessions. The PCA also indicated that flowering traits were more influenced by seed origin while seed traits were affected by flowering spans. Although evaluations were performed in plants during the juvenile phase, accessions TREC 31 and TREC 55 had superior averages for almost all characters evaluated. These results provide a preliminary assessment of the high variability in jatropha accessions evaluated and their potential for use in breeding and genetic improvement programs.     

Development of a sensitive ELISA to quantify apolipoprotein CIII in nonhuman primate serum

Apolipoprotein CIII (apoCIII), a major constituent of triglyceride-rich lipoprotein, has been proposed as a key contributor to hypertriglyceridemia on the basis of its inhibitory effects on lipoprotein lipase. Many immunochemical methods have been developed for human apoCIII quantification, including ELISA. However, a sensitive and quantitative assay for nonhuman primates is not commercially available. We developed a sensitive, quantitative, and highly specific sandwich ELISA to measure apoCIII in both nonhuman primate and human serum. Our assay generates a linear calibration curve from 0.01 μg/ml to 10 μg/ml using an apoCIII standard that was purified from cynomolgus monkey serum. It is highly reproducible (intra- and interplate CV < 5% and < 8%, respectively), sensitive enough to distinguish 10% difference of apoCIII present in serum, and has no interference from purified human apolipoprotein AI, AII, B, CI, CII, or E. The same assay can also be used to measure human apoCIII with a linear calibration curve from 0.005 μg/ml to 1 μg/ml using purified human apoCIII as the standard. This fast and highly sensitive ELISA could be a useful tool to investigate the role of apoCIII in lipoprotein transport and cardiovascular disease.     

The effects of sympathetic stimulation and adenosine on coronary circulation and heart function in diabetes mellitus

The aim of the experiment was to clarify whether the altered coronary reactivity in diabetes mellitus might be caused by a general modification of the sympathetic responses. Six of 12 young mongrel dogs of both sexes were made diabetic with alloxan (560 mmol/kg). This amount of alloxan induced a clinically manifest diabetes, however the animals failed to develop ketosis. The remaining six dogs served as controls. The haemodynamic investigation was performed three months after the induction of diabetes. Under pentobarbital anaesthesia (133 mmol/kg) mean arterial blood pressure, blood flow in the left anterior descending coronary artery, myocardial contractile force of left ventricular wall and heart rate were recorded continuously and the conductivity of coronary arterial bed was calculated during electrical stimulation ( 8V , 1-2-4-8-20 s-1) of the cardiac plexus or during the intracoronary infusion of adenosine (30-60-120-240-480 nmol/kg/min). In alloxan-diabetic dogs electrical stimulation evoked vasoconstriction in the coronary arterial bed, while vasodilation was observed in metabolically healthy animals. The vasodilator effect of adenosine was significantly smaller in diabetic than in control dogs. On the other hand there were no differences either in the alterations of heart rate caused by adenosine or in those of myocardial contractile force induced by adenosine or electric stimulation between the two groups. It is concluded that general alteration of sympathetic responses is not, but rather a modified relation of the receptors to the vessel wall might be responsible for the altered vascular responses in diabetes.     

Stimulus representation in SOP: II. An application to inhibition of delay

The componential extension of SOP accounts for conditioned response (CR) timing in Pavlovian conditioning by assuming that learning accrues with relative independence to stimulus elements that are differentially occasioned during the duration of the conditioned stimulus (CS). SOP, using a competitive learning rule and the assumption that temporal learning emerges via resolution of what is equivalent to an "AX+BX-" discrimination, predicts a progressive increase in the latency of the CR over training, or what Pavlov refer to as "inhibition of delay." Other componential models, which use noncompetitive learning rules, do not predict inhibition of delay. Either type of model makes the prediction indicated, independently of the length of the CS-unconditioned stimulus (US) interval. We report two experiments that demonstrated inhibition of delay when rabbits were trained with relatively long, but not with short, CS-US intervals. To account for this divergence, we assumed that the SOP stimulus trace involves two kinds of elements, some with a temporally distributed pattern of activity over the duration of the CS duration, and some with a randomly distributed pattern. This stimulus representation, not only allows for inhibition of delay with long but not short CS-US intervals, but in combination with SOP's performance rule deduces CR's with "Weber variability."     

Experimental Helicobacter pylori infection induces antral-predominant, chronic active gastritis in hispid cotton rats (Sigmodon hispidus)

BACKGROUND: The hispid cotton rat has proven to be an excellent animal model for a variety of human infectious disease agents. This study was performed to evaluate the use of the cotton rat as a model of Helicobacter pylori infection. MATERIALS AND METHODS: Thirty-eight inbred cotton rats were orogastrically inoculated with a human strain of H. pylori. Twenty-eight control cotton rats were dosed with vehicle only. Animals were sacrificed at 2, 4, 12, 26, or 38 weeks after inoculation for bacterial and histologic and immunologic examinations. RESULTS: Helicobacter pylori was cultured from the glandular stomach of 89% of the infected cotton rats. The level of colonization was consistently high (approximately 10(4-6) colony-forming units/g tissue). Histologically, the spiral bacteria were demonstrated on the epithelial surface and in the foveolae of the gastric mucosa with highest numbers in the antrum. H. pylori infection was associated with antral-predominant, chronic active gastritis which progressively increased in severity over time. By week 26 of infection, moderate antral gastritis had developed with frequent involvement of the submucosa and formation of lymphocytic aggregates. Splenic T cells from infected cotton rats expressed mRNAs for interferon-gamma, interleukin-4, interleukin-6, and interleukin-10 following in vitro stimulation with H. pylori. Serum levels of H. pylori-specific immunoglobulin G were significantly elevated after 12 weeks of infection. CONCLUSIONS: The H. pylori-infected cotton rat represents a novel animal model that should prove useful for studies of H. pylori-induced chronic active gastritis and factors affecting gastric colonization by this pathogen.     

eIF4G and CBP80 share a common origin and similar domain organization: implications for the structure and function of eIF4G

Eukaryotic translation initiation factor 4G (eIF4G) plays a critical role in protein expression, and is at the center of a complex regulatory network. Together with the cap-binding protein eIF4E, it recruits the small ribosomal subunit to the 5'-end of mRNA and promotes the assembly of a functional translation initiation complex, which scans along the mRNA to the translation start codon. Human eIF4G contains three consecutive HEAT domains, as well as long unstructured regions involved in multiple protein-protein interactions. Despite the accumulating data about the structure and function of eIF4G, the mechanisms of coordination and regulation of its interactions with other factors have remained largely unknown. Here, we present evidence that eIF4G and the large subunit of the nuclear cap-binding complex, CBP80, share a common origin and domain structure. We propose that the organization of the individual domains in eIF4G and CBP80 could also be conserved. The structure of CBP80, in complex with the nuclear cap-binding protein CBP20, is used to build a model for the mutual orientation of the domains in eIF4G and their interactions with other factors. The organization of the CBP80-CBP20 complex suggests how the activity of eIF4G in translation initiation could be regulated through a dynamic network of overlapping intra- and intermolecular interactions centered around the eIF4G HEAT domains.     

Vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibres in the penile cavernous tissue of green monkeys (Cercopithecus aethiops)

Summary The cavernous body of green monkeys contains many unmyelinated and few myelinated axons. The unmyelinated axons form terminals in the adventitia of the arteries, between trabecular muscle cells, in the interstitium, and close to endothelium cells of the sinuses. All terminals displayed predominantly small clear vesicles and very few large granular vesicles; small granular vesicles were not seen. However, in rabbit penises, terminals with many large granular vesicles are prominent. Immunohistochemistry (PAP technique) showed a dense network of VIP- and NPY-reactive fibres around the arteries and around trabecular muscles. The density of nerve fibres was particularly high around the subendothelial cushions of the helicine arteries. Double staining for NPY and VIP revealed that both peptides were colocalized. Immunocytochemistry (preembedding PAP technique) showed VIP- and NPY-reactivity in terminals with small clear vesicles; the reaction product was bound to the cytoplasmic face of different membrane types. Although the intracellular localization of the reaction product is probably due to artefactual displacement during preparation, the uniformity of the terminals questions the view that large and small granular vesicles in all species characterize peptidergic and noradrenergic terminals, respectively. The essential findings can be summarized as (1) a high degree of uniformity of nerve terminals, (2) colocalization of VIP and NPY, (3) heavy innervation of the subendothelial cushions of the helicine arteries, and (4) possible innervation of endothelial cells.     

Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.