Fluorescent 2''(3'')-O-aminoacylnucleosides-acceptor substrates for ribosomal peptidyltransferase+.

2'(3')-O-L-Phenylalanylderivatives of fluorescent 1,N6-ethenoadenosine and 3,N4-ethenocytidine were prepared by chemical synthesis. Both compounds are good acceptor substrates in ribosomal peptidyltransferase reactions. Since these compounds cannot form Watson-Crick base pairs, the results indicate that the terminal aminoacyladenosine unit of AA-tRNA is bound to ribosomal protein on the acceptor site of peptidyltransferase and not to rRNA.     

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells

Embryogenic cultures of cultivated carrot ( Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH⁺₄ as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH⁺₄ alone or a combination of NH⁺₄ and NO⁻₃. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.     

Effects of an inhibitor and a mimic of superoxide dismutase on bleomycin mutagenesis in Chinese hamster ovary cells

We have investigated the roles of reactive oxygen species (ROS) in bleomycin (BLM)-induced gene mutations in Chinese hamster ovary (CHO) cells using a superoxide dismutase (SOD) inhibitor, triethylenetetramine (TRIEN), and a SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), to lower and increase intracellular “SOD activity”, respectively. Pretreatment of CHO cells with TRIEN (1 mM) for 1 h enhanced the mutagenic response of BLM (5–50 μg/ml, 1 h treatment) in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO cell clone K1-BH4 (CHO/HPRT assay) and the xanthine-guanine phosphoribosyltransferase (gpt) gene in a CHO-K1 cell derivative AS52 (AS52/GPT assay). Pretreatment with TEMPOL (1 mM) for 1 h decreased the BLM (20–100 μg/ml, 1 h treatment) mutagenicity in the AS52/GPT assay. The mutagenic response of BLM appears to be modulated by the intracellular level of ‘SOD activity’ and hence the intracellular level of ROS. These data provide further evidence for the involvement of ROS in bleomycin mutagenesis in mammalian cells.     

Priming of associative long-term depression in the dentate gyrus by theta frequency synaptic activity.

Associative long-term synaptic depression (LTD) was investigated utilizing negatively correlated activity patterns in the medial and lateral perforant path inputs to the dentate gyrus in anesthetized rats. Normally only nonassociative, or heterosynaptic, LTD is elicited in naive pathways. We report here, however, that associative LTD in the lateral path is readily induced after being "primed" by a brief period of lateral path synaptic activity at a theta rhythm frequency (5 Hz). Priming of associative LTD lasts at least 2 hr and is not seen following priming activity at non-theta frequencies (1 and 15 Hz). N-methyl-D-aspartate receptor activation is critical for establishing the priming effect, but not for the subsequent induction of the associative LTD. These data suggest that theta rhythm activity in the dentate gyrus may predispose the system to a specific form of synaptic plasticity, associative LTD.     

The isotype potential of B cells present in BALB/c mice chronically infected with Mesocestoides corti

Infection of BALB/c mice with Mesocestoides corti results in a chronic infection with a pronounced splenomegaly and hypergammaglobulinemia. A prominent feature of this infection is that the vast majority of serum immunoglobulin produced is restricted to IgG1 and IgM. As much as 30-fold increases in serum IgG1 levels have been noted. To ascertain whether, as a result of infection, the resident B cell pool is committed to IgG1, B cells from infected animals were tested for their ability to produce various isotypes after stimulation. In one series of experiments, B cells from normal and infected animals were used as donor cells in the splenic fragment assay. The results show that the frequency of 2,4-dinitrophenyl-specific and phosphorylcholine-specific B cells remains unaltered in infected animals compared to controls. Importantly, the hapten-specific B cell clones induced were found to express multiple isotypes. These results demonstrate that the nonactivated B cell pool in spleens of infected mice is not committed to IgG1 and IgM production.     

Microanalysis of free and conjugated bile acids by thin-layer chromatography and in situ spectrofluorimetry

A combination of thin-layer chromatography (TLC) and in situ spectrofluorimetry for the determination of free bile acids and bile acids conjugated with glycine or taurine is described. This method makes it possible to determine bile acids concentrations as low as 0.15-0.25 nmol (0.05-0.1 microgram) in a simple and reproducible way. Moreover, information can be obtained about conjugation patterns and relative concentrations of mono-, di-, and trihydroxy bile acids as well as about the presence of abnormal bile acids. After TLC the bile acids are made visible in uv light by dipping the layer in sulfuric acid in diethyl ether and warming it under well-described conditions. The fluorescence of the bile acids on the thin layer can be measured and makes it possible to quantitate them. The method presented here is applicable to bile acid-containing extracts from serum, bile, and feces, and the results are in good agreement with those obtained by enzymatic and gas-liquid chromatographic techniques.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.     

Extravascular circulation in the pituitary of Mugil cephalus (Teleostei)

Summary Extravascular circulation in the pituitary of Mugil cephalus was investigated by injecting live fish with horseradish peroxidase and studying the distribution of the enzyme in the gland. The principal components of the extravascular circulatory system are the pericapillary spaces, and, arising from them, the interlobular and circumhypophyseal spaces. Extensions of these spaces penetrate the glandular parenchyma of the pars distalis, where they merge with pericellular spaces. In the neurohypophysis, pericapillary spaces are connected to the periaxonal spaces.Capillaries penetrating from the proximal neurohypophysis into the pars distalis are accompanied by neurosecretory axons. These axons form a mass of tissue which is limited near the capillaries by the pericapillary spaces and near the adenohypophysis by the interlobular spaces. Toward the interior of the adenohypophysis the amount of nervous tissue accompanying the capillaries progressively diminishes, thus reducing the distance between pericapillary and interlobular spaces. Within the pars distalis, the neurosecretory axons accompanying the capillaries are sparse, and the secretory and stellate cells are mostly located directly adjacent to the pericapillary spaces. In the neuro-intermediate lobe, interlobular spaces outline the neuro-adenohypophyseal boundary.The relationship between extravascular spaces and hormone-secreting cells varies in the different regions of the adenohypophysis depending upon the type of neurosecretory innervation in the respective region. In the regions of prolactin and gonadotropin cells, where neurosecretory axons are in direct contact with the secretory cells, the hormone-secreting and stellate cells are adjacent to the pericapillary spaces. In the regions of ACTH and STH cells, secretory and stellate cells are found adjacent to the interlobular spaces, which are interposed between the cells and the neurosecretory axons.Abbreviations AH adenohypophysis - CH circumhypophyseal - DNH distal neurohypophysis - HRP horseradish peroxidase - NH neurohypophysis - NS neurosecretory - PD pars distalis - PI pars intermedia - PPD proximal pars distalis - RNH rostral neurohypophysis - RPD rostral pars distalis This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS Geesthacht-Tesperhude, Federal Republic of Germany     

Evidence for restricted oligosaccharide mobility at the erythrocyte membrane surface: A fluorescence study

The glycophorins of whole, human erythrocytes were labeled at their sialic acid residues with one of three fluorescent probes. After preparation of the erythrocyte ghosts, the mobility of each fluorescent probe on the intact membrane was compared with its mobility on the isolated, labeled glycopeptides dissolved in aqueous buffer. A four- to ninefold decrease in the rotational relaxation time, as defined by the Perrin equation, accompanied the proteolytic removal of the labeled glycopeptides from the membrane. This suggests that the fluorescent probes, and by extrapolation, the sugars to which they are immediately attached, are restricted in their mobility at the membrane surface. A crude model of the carbohydrate layer of the erythrocyte surface was constructed by incorporating the labeled, tryptic glycopeptides into agarose gels of different agarose content. A decrease in the probe's mobility was observed as agarose content was raised. This indicates that the high oligosaccharide density at the erythrocyte membrane surface may contribute to the observed immobilization of the fluorescent probes in situ.