5-Chlorodeoxyuridine, more effectively than 5-bromodeoxyuridine, induces the formation of specific dicentric chromosomes in cells with micronuclei

5-chlorodeoxyuridine (5-ChldU) and 5-bromodeoxyuridine (5-BrdU) were tested in respect of the formation of specific dicentric chromosomes in cells with micronuclei. It is shown that the frequency of dicentrics is higher when 5-ChldU is used. On the ground of results of this study and of the previous data it is concluded that the activity of halogenated deoxyuridines is falling with molecular weight increasing.     

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P<.02), compared to control cells. Both EPA and DHA reduced TNF-alpha, IL-1beta and IL-6 mRNA expression. In all cases, the effect of DHA was significantly more potent than that of EPA (P<.01). Furthermore, a low dose (25 microM) of DHA had a greater inhibitory effect than that of EPA on macrophage IL-1beta (P<.01 and P<.04, respectively) and IL-6 (P<.003 and P<.003, respectively) production following 0.01 and 0.1 microg/ml LPS stimulation. Both EPA and DHA down-regulated LPS-induced NF-kappaB/DNA binding in THP-1 macrophages by approximately 13% (P< or =.03). DHA significantly decreased macrophage nuclear p65 expression (P< or =.05) and increased cytoplasmic IkappaBalpha expression (P< or =.05). Although similar trends were observed with EPA, they were not significant. Our findings suggest that DHA may be more effective than EPA in alleviating LPS-induced pro-inflammatory cytokine production in macrophages - an effect that may be partly mediated by NF-kappaB. Further work is required to elucidate additional divergent mechanisms to account for apparent differences between EPA and DHA.     

LARGE2 facilitates the maturation of alpha-dystroglycan more effectively than LARGE

The LARGE gene is thought to encode a putative glycosyltransferase because of its typical topology. However, no enzyme activity has been demonstrated yet, although the gene apparently supports the functional maturation of alpha-dystroglycan by glycosylation when it is transfected into cells. A novel homologous gene to LARGE was identified and named LARGE2. LARGE2 recombinant was co-expressed with alpha-dystroglycan in human embryonic kidney 293T cells to determine its activity to support the maturation of alpha-dystroglycan. The alpha-dystroglycan co-transfected with LARGE2 was more highly glycosylated than that co-transfected with LARGE. Pull-down experiments demonstrated binding activity of LARGE2 as well as LARGE toward alpha-dystroglycan. LARGE2 was found to support the maturation of alpha-dystroglycan more effectively than LARGE. Both of them are ubiquitously expressed in many tissues, except the brain where LARGE2 was not expressed at all. This compensatory function can explain the residual functionally glycosylated alpha-dystroglycan in a patient with MDC1D whose LARGE genes are congenitally null.     

Enhancement of shoot regeneration by treatment with inhibitors of auxin biosynthesis and transport during callus induction in tissue culture of Arabidopsis thaliana

In two-step culture systems for efficient shoot regeneration, explants are first cultured on auxin-rich callus-inducing medium (CIM), where cells are activated to proliferate and form calli containing root-apical meristem (RAM)-type stem cells and stem cell niche, and then cultured on cytokinin-rich shoot-inducing medium (SIM), where stem cells and stem cell niche of the shoot apical meristem (SAM) are established eventually leading to shoot regeneration. In the present study, we examined the effects of inhibitors of auxin biosynthesis and polar transport in the two-step shoot regeneration culture of Arabidopsis and found that, when they were applied during CIM culture, although callus growth was repressed, shoot regeneration in the subsequent SIM culture was significantly increased. The regeneration-stimulating effect of the auxin biosynthesis inhibitor was not linked with the reduction in the endogenous indole-3-acetic acid (IAA) level. Expression of the auxin-responsive reporter indicated that auxin response was more uniform and even stronger in the explants cultured on CIM with the inhibitors than in the control explants. These results suggested that the shoot regeneration competence of calli was enhanced somehow by the perturbation of the endogenous auxin dynamics, which we discuss in terms of the transformability between RAM and SAM stem cell niches.     

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.     

The effect of chloramphenicol on the growth and xylogenesis in callus ofHaplopappus gradlis

The effect of chloramphenicol (CP) on the differentiation of callus cells ofHaplopappus gracilis into tracheary elements (TE) was studied. CP (1 mg l^-1) added to the medium stimulating the differentiation was shown to have an inhibitory effect. This observation points to the importance of the impaired functions of mitochondria in the processes leading to the differentiation of callus cells into TE.     

Dose dependent treatment with isotretinoin induces more changes in the ileum than in the duodenum and jejunum in Wistar rats

Acne is the most common skin disorder and can directly affect the patients’ self-esteem. Systemic treatment has been indicated for nodular, cystic or persistent acne rather than another type of treatment, such as a topic one. Isotretinoin is an analogue of vitamin A and by suppressing the sebaceous glands the disease can be controlled. This study was designed to mimic the treatment performed in young patients using the dosage of 1 mg/kg, and a higher one of 10 mg/kg, for 60 days in young male Wistar rats. 24 Wistar rats were divided into four groups: control(water), D0(soybean oil, control group), D1(1 mg/kg of Isotretinoin solution), D10(10 mg/kg of Isotretinoin solution). Using the morphometry tool and histochemical techniques we evaluated the villus, intestinal crypts, and goblet cells to find signs of possible alterations of the duodenum, jejunum and ileum segments of the small intestine. We found no signs of changes in the jejunum mucosa after 60 days of treatment with 1 mg/kg and 10 mg/kg. The duodenum is also less affected, whereas significant modifications were found in the ileum. The goblet cell frequency was altered, indicating a proliferative potential for the substance. Although some patients have described intestinal symptoms, no important alterations were found with this protocol, reaffirming the security involved in the treatment with this substance.     

Effects of auxin on the spatial distribution of cell division and xylogenesis in lettuce pith explants

Summary Cylinders of pith parenchyma were tissue-cultured with their opposite ends on media which differed only in content of the morphogens auxin (IAA), sucrose, or zeatin. A range of concentrations of each of these morphogens applied at one end (none at the other end) resulted in distribution patterns of cell division and xylogenesis that were attributable to interaction between inductive levels and morphogen mobility. Auxin was crucial for tracheary patterns: large tracheary elements formed by direct differentiation of pith cells near the auxin source, smaller but still roughly isodiametric tracheary elements formed after cell division, and tracheary strands developed where, presumably, auxin transport had become polarized and then canalized. Xylogenesis was confined to regions within millimeters of the auxin source, and [¹⁴C]IAA studies showed a steep logarithmic concentration gradient along the cylinder. Patterns of tracheary strands and rings revealed that the pith explants retained some polarity from the stem from which they had been excised. However, the direction of flow of applied auxin was more effective than original polarity in controlling the orientation of tracheary strands and their constituent tracheary elements. It seems that, in tissues with little or no polarity, diffusive flow of auxin gradually induces polar flow in the same direction, together with an associated bioelectric current, and that this orients the cortical microtubules that in turn determine the orientations of cell elongation and of the secondary wall banding in tracheary elements.Abbreviations IAA indoleacetic acid - NAA naphthaleneacetic acid - TIBA triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Distribution of linker histone variants during plant cell differentiation in the developmental zones of the maize root, dedifferentiation in callus culture after auxin treatment

Although several linker histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study, we identified linker histone variants by using specific anti-histone Hl antibodies. Each variant's ratio to total Hl in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them was estimated in order to define possible alterations either during plant cell differentiation or during their dedifferentiation. We also evaluated linker histone variants' ratios in the developmental zones of maize roots treated with auxin in order to examine the effects of exogenous applied auxin to linker histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing each variant and correlate them with the physiological status of the plant cells. According to the results presented in this study, linker histone variants' ratios are altered in the developmental zones of maize root, while they are similar to the meristematic zone in samples from callus cultures and to the differentiation zone in samples from roots treated with auxin. We propose that the alterations in linker histone variants' ratios are correlated with plant cell differentiation and dedifferentiation.     

Participation of auxin and abscisic acid in the regulation of seasonal variations in cambial activity and xylogenesis

Summary The current notion that hormonal level and cell response are clearly correlated has often been challenged recently. During the period of cambial activity, auxin content seems to control the intensity of mitosis and some features of the resulting wood, but not the duration of the active period itself. During cambial rest, the indole-3-acetic acid (IAA) level often remains high in the cambium, but the cell sensitivity to auxin is low. The decrease of auxin transport in autumn is sometimes interpreted as a major qualitative change affecting the pattern of transport, and sometimes as a secondary change occurring later than rest onset. The causes of the seasonal variation of cambial response remain unknown. A hypothesis is proposed that accounts for the structural-functional changes occurring in cambial cells during the onset of dormancy. Abscisic acid (ABA) may reduce wood production and xylem cell enlargement in late summer. An important amount of ABA may be present in the cambial zone in autumn after drought stress and in spring in the young growing shoot. Changes in ABA level do not appear to be clearly correlated with the different steps of cambial rest and activity. Beyond the role of ABA as a stress mediator, its participation in the annual regulation of cambial activity remains unclear. Its distribution in the most alkaline compartments may account for the particularities of its seasonal activity. The involvement of IAA and ABA in cambial growth is discussed within the scope of a possible annual alternation of two different metabolisms in the cambial cell.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA gibberellic acid - GC-MS gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PA phaseic acid - RNA ribonucleic acid - SICM single ion current monitoring - SIM selected ion monitoring     

Ovine fetal development is more sensitive to perturbation by the presence of serum in embryo culture before rather than after compaction

The effects on subsequent fetal development of the presence or absence of serum at different times during IVC of ovine zygotes were studied. Zygotes, recovered from superovulated ewes 36h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media supplemented with either BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). Serum was present or absent during the first two and last 2 days of IVC giving four treatments (SOF-/SOF-; SOF-/SOF+;SOF+/SOF- and SOF+/SOF+). In total, 224 embryos, including 26 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Presence of serum during IVC had a biphasic effect on embryo development. The inclusion of serum during the first 2 days of IVC retarded early embryo development while the inclusion of serum during the last 2 days of IVC produced more blastocysts by day 6. These effects were independent of each other. The presence of serum during the first 2 days of IVC resulted in increased weights of gravid uterus, placenta, fetus, fetal heart and liver. The incidence of fetuses whose total or organ weights were greater than three standard deviations above the corresponding mean weights of control fetuses was also greater when serum was present during the first 2 days of IVC. However, even when serum was absent throughout IVC there was still an infrequent incidence of fetal weights greater than three standard deviations above the mean for control fetuses. These observations provide evidence that it is the early pre-compaction stages of embryo development that are particularly sensitive to perturbations leading to abnormal fetal development.     

Inhibition of growth and xylogenesis and promotion of vacuolation in prunus callus by the flavanone prunin

Callus tissue of Prunus avium L. responded to supplied prunin (naringen in 7-glucoside) showing vaculoation and storage of oligomeric proanthocyanidins. In addition, prunin caused restricted callus initiation and/or less callus growth. When prunin was omitted from the medium numerous tracheids and more peroxidases were formed in the callus.Abbreviations DAB diaminobenzidine - DMACA p-diaminocinnamaldehyde - DWG dihydrowogonine 7-glucoside - P prunin - N naringenin     

Changes in auxin protectors and IAA oxidases during the rooting of chestnut shoots in vitro

Auxin protectors and IAA oxidase activity were comparatively analyzed in the upper and the lower parts of shoots of chestnut ( Castanea sativa Mill.) cultivated in vitro with indolebutyric acid (IBA) pretreatment. Rhizogenesis of the shoots is accompanied by an increase in auxin protectors in the lower parts and by a decrease of these protectors in the upper parts. Besides, the IAA oxidase activity declines in the basal parts during the rooting process while it increases in the upper ones. These biochemical events would enhance the IAA level in the rooting region of the shoots. In untreated, non-rooted cuttings, the IAA oxidase activity remains low in the upper parts and high in the basal parts of the shoots. The results thus indicate that the IBA treatment may control the endogenous auxin level of the cuttings, either through a direct regulation of the IAA oxidase system or more indirectly through the transport of auxin protectors.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

Improvement of rice callus regeneration in the presence of NaCl

The effects of abscisic acid (37.8 μM), polyethylene glycol (5%), proline (10 mM), tryptophan (490 μM) and indoleacetic acid (5.7 μM) on rice callus regeneration were studied at various doses of NaCl (0, 50 and 100 mM) on three month-old mature embryo-derived callus of two japonica (I Kong Pao and Aiwu) and two indica (IR 2153 and Nona Bokra) rice cultivars differing in salinity tolerance. NaCl strongly decreased the regeneration frequency of all cultivars but slightly increased the survival of regenerated plantlets. Tryptophan stimulated regeneration and increased subsequent survival rates of regenerated plantlets in all cultivars at all NaCl doses. Abscisic acid and polyethylene glycol, though not affecting the final regeneration percentages, delayed regeneration and reduced the mean number of plantlets produced per regenerating callus in all cultivars, as well as rooting ability and survival of regenerated plantlets in indica genotypes. Proline had no marked effect on regeneration, whatever the NaCl dose or cultivar, while indoleacetic acid reduced shoot regeneration and increased root regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.