Molecular phylogeny and host-plant use (Lamiaceae) of the Thymogethes pollen beetles (Coleoptera)

The 24 members of the Euro-Asiatic genus Thymogethes are highly specialized pollen beetles associated as larvae with flowers of Lamiaceae Nepetoideae. All members of the genus were analysed in within the framework of an integrative taxonomy approach, which was aimed to reconstruct the phylogenetic relationships, as well as the possible pattern of evolution of their larval-host-plant association. Evidence from multiple molecular markers [COI; 16S; H3], combined with an estimation of divergence times using an average rate of 0.0177 substitutions/site/My among branches, placed the origin of the genus at a minimum of 9–10 Mya. This date of origin approximates the known evolution of the host plants in Euro-Mediterranean areas. Evidence from combined molecular and cladistic morphological analyses resulted in suitable agreement with the previously established morphology-based systematics of the genus, although members of the exilis species-group were split into three clades. The only disagreement between results of this new combined phylogeny and previous classification is in the exclusion of “Thymogethes” grenieri. This species is herein positioned outside the genus, based on molecular evidence. Our analysis depicts several Thymogethes species differentiating in the last few Mys, specifically those included in the T. lugubris species-group. Combined evidence from DNA, morphology and ancestral state parsimony reconstruction of larval-host-plant associations suggests that subtribe Menthinae likely represents the ancestral host plants, with a series of independent host shifts during the radiation of the clade, in association first with Menthinae and subsequently with Lavandulinae and Nepetinae. Steno-oligophagy is the most frequent (86%) condition, while strictly monophagous species are less numerous (14%).     

Germ line configuration of the BCL-2 major breakpoint region in hyperplastic lymph nodes.

The configuration of the BCL-2 major breakpoint region was analyzed by Southern blot hybridization and polymerase chain reaction amplification in DNA derived from 44 benign hyperplastic lymph nodes with follicular overgrowth. None of the cases exhibited translocation of the BCL-2 gene at the major breakpoint region of chromosome 18. The potential usefulness of molecular genetic detection of BCL-2 translocation in the differential diagnosis between hyperplastic lymph nodes and lymphomas is suggested.     

Liver autofluorescence properties in animal model under altered nutritional conditions

Autofluorescence spectroscopy is a promising and powerful approach for an in vivo, real time characterization of liver functional properties. In this work, preliminary results on the dependence of liver autofluorescence parameters on the nutritional status are reported, with particular attention to vitamin A and lipid accumulation in liver tissue. Normally fed and 24 h starving rats were used as animal models. Histochemical and autofluorescence analysis showed that lipids and vitamin A colocalize in the liver parenchyma. Fasting condition results in a parallel increase in both lipids and vitamin A. Autofluorescence imaging and microspectrofluorometric analysis carried out on unfixed, unstained tissue sections under 366 nm excitation, evidenced differences in both spectral shape and response to continuous irradiation between liver biopsies from fed and starving rats. As to photobleaching, in particular, fitting analysis evidenced a reduction of about 85% of the signal attributable solely to vitamin A during the first 10 s of irradiation. The tissue whole emission measured in fed and starving rat livers exhibited reductions of about 35% and 52%, respectively, that are closely related to vitamin A contents. The findings open interesting perspectives for the set up of an in situ, real time diagnostic procedure for the assessment of liver lipid accumulation, exploiting the photophysical properties of vitamin A.     

Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.     

The major antenna complex of photosystem II has a xanthophyll binding site not involved in light harvesting.

We have characterized a xanthophyll binding site, called V1, in the major light harvesting complex of photosystem II, distinct from the three tightly binding sites previously described as L1, L2, and N1. Xanthophyll binding to the V1 site can be preserved upon solubilization of the chloroplast membranes with the mild detergent dodecyl-alpha-d-maltoside, while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2, and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesized zeaxanthin, which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.     

Conformational Analysis of a Synthetic Antimicrobial Peptide in Water and Membrane-Mimicking Solvents: A Molecular Dynamics Simulation Study

We have investigated structural and dynamic properties of the synthetic peptide hlF1-11 (GRRRSVQWCA, i.e., the first 11 N-terminal amino acids of the human lactoferrin protein) in water, 250 mM NaCl solution, 50% (V/V) water–trifluoroethanol mixture, and in the membrane mimetic 4:4:1 methanol–chloroform–water mixture. For comparison, we have also performed analogous simulations for the biologically inactive control peptide featuring Ala substitutions in the 2, 3, 6 and 9 positions of the hlF1-11 sequence. Statistical analyses of the trajectories indicate that only in the membrane-mimicking medium hlF1-11 adopts preferentially a conformation suitable to interact effectively with the membrane. In this conformation the peptide cationic region is rather flexible and elongated, while the C-terminal hydrophobic moiety appears as a more rigid hairpin-shaped loop approximately perpendicular to the cationic region. No such conformation is statistically relevant for the control peptide.     

Tissue preference and differentiation of malignant rat x mouse hybrid cells in chimaeric mouse fetuses.

Mouse teratocarcinoma cells (OTT6050) deficient for thymidine kinase were fused with rat hepatoma cells ( Fu5AH ) deficient for hypoxanthine phosphoribosyltransferase using inactivated Sendai virus. The hybrid cells were selected and cultured in the presence of HAT medium. A clonally established hybrid cell line ( As3 ), which in addition to its mouse genome contains several rat chromosomes, expresses rat specific enzyme variants and produces large primarily undifferentiated tumors, with some hepatoma characteristics in athymic nude mice. To reveal the in vivo developmental potential of these cells and to determine whether, under different experimental conditions, they are capable of participating in tissue differentiation, the As3 cells were injected into mouse blastocysts from the C57BL/6 strain. The experimental blastocysts were then transferred into the uteri of pseudopregnant foster mothers to allow further development. From a total of 212 blastocysts transplanted, 61 fetuses developed and were analysed for As3 contributions between the 10th and 18th day of gestation. Four fetuses at day 18 showed hybrid cell participation in their livers and a few organs of only endo-mesodermal origin, as judged from the presence of rat-specific enzyme variants. The enzymes were organ-specifically expressed (e.g., lactate dehydrogenase) or appeared newly during in situ differentiation while being absent in the original hybrid cells (e.g., glycerol-3-phosphate dehydrogenase). During short in vitro culture of the chimaeric organs, it was possible to select for the hybrid cells which reverted to an enzyme pattern simiar to but not identical with the As3 cell line and different to that observed in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions.

The contribution of endogenous fluorophores - such as proteins, bound and free NAD(P)H, flavins, vitamin A, arachidonic acid - to the liver autofluorescence was studied on tissue homogenate extracts and on isolated hepatocytes by means of spectrofluorometric analysis. Autofluorescence spectral analysis was then applied to investigate the response of single living hepatocytes to experimental conditions resembling the various phases of the organ transplantation. The following conditions were considered: 1 h after cells isolation (reference condition); cold hypoxia; rewarming-reoxygenation after cold preservation. The main alterations occurred for NAD(P)H and flavins, the coenzymes strictly involved in energetic metabolism. During cold hypoxia NAD(P)H, mainly the bound form, showed an increase followed by a slow decrease, in agreement with the inability of the respiratory chain to reoxidize the coenzyme, and a subsequent NADH reoxidation through alternative anaerobic metabolic pathways. Both bound/free NAD(P)H and total NAD(P)H/flavin ratio values were altered during cold hypoxia, but approached the reference condition values after rewarming-reoxygenation, indicating the cells capability to restore the basal redox equilibrium. A decrease of arachidonic acid and vitamin A contributions occurred after cold hypoxia: in the former case it may depend on the balance between deacylation and reacylation of fatty acids, in the latter it might be related to the vitamin A antioxidant role. An influence of physico-chemical status and microenvironment on the fluorescence efficiency of these fluorophores cannot be excluded. In general, all the changes observed for cell autofluorescence properties were consistent with the complex metabolic pathways providing for energy supply.