Isolation of a single messenger RNA and of the corresponding gene in Plasmodium berghei

A genomic library of Plasmodium berghei DNA was constructed using lambda 47.I as a vector. It represents 90% of Plasmodium genome. Genes expressed during the intraerythrocytic stage of P. berghei were isolated among the recombinant clones of the library using labelled cDNA complementary to the polyA + Plasmodium mRNA extracted during this stage. The purified coding strand of an expressed clone was utilized to catch the corresponding mRNA(s). The hybridized mRNA fraction was eluted and in vitro translated. Translation products were analyzed by gel electrophoresis; the gel fluorography revealed a single protein band of 32.500 daltons of molecular weight, corresponding to a 900bp coding region in the examined clone.     

Swordfish (Xiphias gladius Linnaeus 1758) harpoon fishery: a method of evaluation of swordfish presence in the Strait of Messina (Central Mediterranean Sea)

The objective of this study was to examine the space and environment variables that contributed to the presence of swordfish in the Strait of Messina during the 2003 and 2004 harpoon fishing activity. The relationships between catches per location and environmental factors (current, moon phase and sea surface temperature) were analyzed using a generalized linear model (GLM). The GLM analysis showed differences in CPUE (catch per unit effort) values in terms of swordfish numbers per location over the 2‐year period. The central locations of the Strait were found to be teeming with fish; the CPUE was also higher in these areas during the new and rising moon phases.     

Morphology and distribution of chromaffin cells in the adrenal gland of cordylidae (Reptilia,Sauria): A comparative study

The distribution of the adrenaline and noradrenaline chromaffin cells in the adrenal glands of 10 members of the family Cordylidae have been examined. In the genus Gerrhosaurus, all the catecholamine cells lie on the surface of the adrenal gland, forming a continuous envelope of one or two layers of cells that mainly contain noradrenaline (NA). In the genus Platysaurus, the chromaffin envelope is intermittent. There are relatively large tracts of interspersed interrenal tissue containing some adrenaline cells (A). Islets of chromaffin cells are scattered between these interrenal tracts. In the genus Pseudocordylus and the genus Cordylus, the superficial chromaffin cells tend to gather into a multilayered dorsal mass, containing mainly NA cells. Inside the interrenal parenchyma, there are always numerous chromaffin islets, containing mainly A cells.     

Polyamines: fundamental characters in chemistry and biology

Polyamines are small cationic molecules required for cellular proliferation and are detected at higher concentrations in most tumour tissues, compared to normal tissues. Agmatine (AGM), a biogenic amine, is able to arrest proliferation in cell lines by depleting intracellular polyamine levels. It enters mammalian cells via the polyamine transport system. Agmatine is able to induce oxidative stress in mitochondria at low concentrations (10 or 100 μM), while at higher concentrations (e.g. 1–2 mM) it does not affect mitochondrial respiration and is ineffective in inducing any oxidative stress. As this effect is strictly correlated with the mitochondrial permeability transition induction and the triggering of the pro-apoptotic pathway, AGM may be considered as a regulator of this type of cell death. Furthermore, polyamine transport is positively correlated with the rate of cellular proliferation. By increasing the expression of antizyme, a protein that inhibits polyamine biosynthesis and transport, AGM also exhibits a regulatory effect on cell proliferation. Methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosyl-l-methionine decarboxylase, displaying anticancer activity, is a structural analogue of the natural polyamine spermidine. MGBG has been extensively studied, preclinically as well as clinically, and its anticancer activity has been attributed to the inhibition of polyamine biosynthesis and also to its effect on mitochondrial function. Numerous findings have suggested that MGBG might be used as a chemotherapeutic agent against cancer.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin.

7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound structurally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6. AzMC was synthesized and utilized to label the substrate binding site of UGT1A6. AzMC exhibits a fluorescence spectrum with maximum excitation and emission wavelengths of 380 and 442 nm, respectively. Upon irradiation, the probe irreversibly inhibited glucuronidation activity measured with para-nitrophenol (pNP) as substrate and interacted with UGT1A6 according to a saturable process indicative of reversible binding before covalent incorporation of the photoaffinity label. This inhibition was both time and concentration dependent and led to the calculation of an inhibition constant, k(2) = 0.113 mM min(-1), and dissociation constant, K(d) = 2.89 mM, for the reaction. Partial photoinactivation of UGT1A6 with AzMC revealed that the probe decreased the apparent V(max) of the pNP glucuronidation reaction, but not the K(m). Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate for the enzyme, or by preincubation with an active-site directed inhibitor, 5'-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-su lfonyl]-2 ',3'-O-isopropylideneuridine. In contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affect the photoaffinity labeling of UGT1A6 by 5-[beta-(32)P]N(3)UDP-GlcUA, a photoaffinity analog of UDP-GlcUA. Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Collectively, these results strongly indicate that AzMC specifically binds to the UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding proteins revealed a conserved motif, YXXXKXXPXP. It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins.     

Metastatic tumour cells favour the generation of a tolerogenic milieu in tumour draining lymph node in patients with early cervical cancer

Objective  We compared the immune system state in metastatic tumour draining lymph nodes (mTDLN) and metastasis free TDLN (mfTDLN) in 53 early stage cervical cancer patients to assess whether the presence of metastatic tumour cells worsen the balance between an efficacious anti-tumour and a tolerogenic microenvironment. Methods  The immune system state was measured by immunophenotypic and functional assessment of suppressor and effector immune cell subsets. Results  Compared to mfTDLN, mTDLN were significantly enriched in CD4⁺Foxp3⁺ regulatory T cells (Treg), which, in addition, exhibited an activated phenotype (HLA-DR⁺ and CD69⁺). Treg in mTDLN were also significantly enriched in neuropilin-1 (Nrp1) expressing cells, a subset particularly potent in dampening T cell responses. mTDLN tended to be enriched in a population of CD8⁺Foxp3⁺T cells (operationally defined as CD8⁺Treg) that showed a suppressor potency similar to Treg under the same experimental conditions. Plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) generally show distinct roles in inducing T cell tolerance and activation, respectively. In line with the excess of suppressor T cells, the ratio pDC to mDC was significantly increased in mTDLN. Immunohistochemical testing showed that metastatic tumour cells produced the vascular endothelial growth factor, a natural ligand for Nrp1 expressed on the cell surface of Nrp1⁺Treg and pDC, and therefore a potential mediator by which tumour cells foster immune privilege in mTDLN. Consistent with the overall tolerogenic profile, mTDLN showed a significant Tc2 polarisation and tended to contain lower numbers of CD45RA⁺CD27⁻ effector memory CD8⁺T cells. Conclusions  The increased recruitment of suppressor type cells concomitant with the scarcity of cytotoxic type cells suggests that in mTDLN the presence of tumour cells could tip the balance against anti-tumour immune response facilitating the survival of metastatic tumour cells and possibly contributing to systemic tolerance.