Correlation of sequential floral and male gametophyte development and preliminary results on anther culture in <Emphasis Type="Italic">Opuntia ficus-indica</Emphasis>

Before approaching anther culture as a tool to trigger an androgenic response in a new species, it is advisable to characterize and correlate flower and male gametophyte development to enable reproducible identification of the appropriate starting material. Buds and flowers of Opuntia ficus-indica cv. Gialla were classified in eight stages according to their total length at the earlier stages and the length of the corolla in flowers with emerging sepals. Due to the low condensation of chromatin in the microspore nucleus as well as in the vegetative nucleus of the bi- and tricellular pollen along with the high autofluorescence of the intricate exine, DAPI staining turned out not to be feasible in this species. Therefore an approach based on light-microscopy observation of semithin sections was used. These sections were stained with toluidine blue for general structure recognition and I₂KI to study starch deposition. Correlations were made between the sequential floral and male gametophyte development. Using this approach we determined the timing of pollen formation and observed that pollen development is impaired in plants producing seedless fruits. Furthermore, anther culture was carried out with anthers collected from flower buds at stages 2 and 3. Most of the anthers produced callus, however no regeneration was obtained.     

Role of plasminogen in propagation of scrapie

To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg(-/-)), heterozygous plasminogen-deficient (Plg(+/-)), and wild-type (Plg(+/+)) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrP(Sc)). Only after i.p. inoculation, a slight, although significant, difference in survival (P < 0.05) between Plg(-/-) and Plg(+/+) mice was observed. Neuropathological examination and Western blot analysis were carried out when the first signs of disease appeared in Plg(+/+) animals (175 days after i.p. inoculation) and when mice reached the terminal stage of illness. At the onset of symptoms, PrP(Sc) accumulation was higher in the brain and spleen of Plg(+/+) and Plg(+/-) mice than in those of Plg(-/-) mice, and these differences were paralleled by differences in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures. Immunohistochemical analysis of the spleens before inoculation did not show any impairment of the immune system affecting follicular dendritic or lymphoid cells in Plg(-/-) mice. Once the disease progressed and mice began to die of infection, differences were no longer apparent in either brains or spleens. In conclusion, our data indicate that plasminogen has no major effect on the survival of scrapie agent-infected mice.     

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.     

O-Methylglucogalloyl esters: synthesis and evaluation of their antimycotic activity

The two anomers of O-methyl gluco-2,3-digalloyl esters were synthesized and their antimycotic activity toward yeasts of biomedical importance was evaluated. When used at subinhibitory concentration and regardless of stereochemistry at the anomeric carbon, these compounds enhance the antimycotic activity of Amphotericin B.     

Rbm19 is a nucleolar protein expressed in crypt/progenitor cells of the intestinal epithelium

Intestinal development and homeostasis rely on the coordination of proliferation and differentiation of the epithelium. To better understand this process, we are studying Rbm19, a gene expressed in the gut epithelium that is essential for intestinal morphogenesis and differentiation in the zebrafish (Development 130, 3917). Here we analyzed the expression of Rbm19 in several biological contexts that feature proliferation/differentiation cell fate decisions. In the undifferentiated embryonic gut tube, Rbm19 is expressed throughout the epithelium, but then becomes localized to the crypts of Lieberkühn of the adult intestine. Consistent with its expression in adult crypt/progenitor cells, expression is widespread in human colorectal carcinomas and dividing Caco-2 cells. Its expression in Caco-2 cells recapitulates the in vivo pattern, declining when the cells undergo confluence-induced arrest and differentiation. Rbm19 protein localizes to the nucleolus during interphase and to the perichromosomal sheath during mitosis, in accordance with the pattern described for other nucleolar proteins implicated in ribosome biogenesis. Interestingly, the loss of nucleolar rbm19, nucleolin/C23, and nucleophosmin/B23 in confluent Caco-2 cells did not signify loss of nucleoli as detected by electron microscopy. Taken together, these data point to the nucleolus as a possible locus for regulating the proliferation/differentiation cell fate decision in the intestinal epithelium.     

Effect of indinavir used alone or in double or triple combination with AZT and ddC on human immune functions

Indinavir (IDV) is a potent and selective human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) widely used in antiretroviral therapy, but its effects on the immune system are relatively unknown. In this study we have investigated the in vitro effect of IDV on normal human peripheral blood mononuclear cells (PBMC). We used the drug alone or in double and triple combination with AZT and ddC to assess whether IDV interferes with the previously observed immunomodulatory effects induced by AZT and ddC. We found that proliferative response, induction of immunoglobulins (Ig) production and cytokine production was not modulated by IDV. More importantly, IDV used in double or triple combination with AZT and ddC, does not further strenghten the inhibition of proliferative response induced by AZT and is able to abrogate the inhibitory effect induced by ddC on proliferative response. Similarly, IDV/AZT, IDV/ddC and IDV/AZT/ddC combinations does not strenghten the modulation of TNF-alpha, IFN-gamma and IL-4 induced by AZT, ddC and AZT/ddC. On the other hand, IDV neutralizes the up-regulating effects of AZT on IL-2 production while the up-regulating effects of ddC on IL-2 production is not affected. These data suggest that IDV used in combination with AZT and ddC did not add any further immunotoxicity.     

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography

The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias <15%, except at the lowest limit of quantification (<20%). The inter-assay coefficients of variation and bias were <15%. The application of this method to plasma and urine samples of five CYP2D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.     

Oxytocin induces proliferation and migration in immortalized human dermal microvascular endothelial cells and human breast tumor-derived endothelial cells

Oxytocin either increases or inhibits cell growth in different cell subtypes. We tested here the effect of oxytocin on cell proliferation and migration of human dermal microvascular endothelial cells (HMEC) and tumor-associated endothelial cells purified from human breast carcinomas (B-TEC). Oxytocin receptors were expressed in both cell subtypes at mRNA and protein levels. Through oxytocin receptor, oxytocin (1 nmol/L-1 mumol/L) significantly increased cell proliferation and migration in both HMEC and B-TEC, and addition of a selective oxytocin antagonist fully reverted these effects. To verify whether a different expression of adhesion molecule-related genes could be responsible for the oxytocin-induced cell migration, untreated and treated cells were compared applying a microarray technique. In HMEC, oxytocin induced the overexpression of the matrix metalloproteinase (MMP)-17, cathepsin D, and integrin beta(6) genes. In B-TEC, oxytocin significantly switched on the gene profile of some MMP (MMP-11 and MMP-26) and of integrin beta(6). The up-regulation of the integrin beta(6) gene could be involved in the oxytocin-induced cell growth, because this subunit is known to determine activation of mitogen-activated protein kinase-extracellular signal-regulated kinase 2, which is involved in the oxytocin mitogenic effect. In B-TEC, oxytocin also increased the expression of caveolin-1 at gene and protein levels. Because oxytocin receptor localization within caveolin-1-enriched membrane domains is necessary for activation of the proliferative (instead of the inhibitory) response to oxytocin, its enhanced expression can be involved in the oxytocin-induced B-TEC growth as well. Altogether, these data indicate that oxytocin contributes to cell motility and growth in HMEC and B-TEC.     

Effect of polyamines on in vitro anther culture of Citrus clementina Hort. ex Tan

The improvement of the induction rate in Citrus anther culture is important for taking practical advantage of the haploid potential in breeding. The influence of polyamines on anther culture of Citrus clementina, cv Nules, with particular attention to the free, soluble and insoluble-conjugated polyamine levels, has been investigated. Putrescine, spermidine and putrescine plus spermidine, were added to the standard induction medium. Before culture, spermidine was the most abundant among the free polyamines detected in anthers. The exogenous supply of either putrescine or spermidine, either independently or combined, effected greater uptake and accumulation of polyamines. The addition of 2 mM spermidine to the medium stimulated gametic embryogenesis in clementine Nules, whereas putrescine did not influence embryo production. Regenerants were mostly tri-haploids; a few doubled-haploids and no haploid plants were obtained.