Three new small nucleolar RNAs that are psoralen cross-linked in vivo to unique regions of pre-rRNA.

We have recently described three novel human small nucleolar RNA species with unique nucleotide sequences, which were named E1, E2, and E3. The present article describes specific psoralen photocross-linking in whole HeLa cells of E1, E2, and E3 RNAs to nucleolar pre-rRNA. These small RNAs were cross-linked to different sections of pre-rRNA. E1 RNA was cross-linked to two segments of nucleolar pre-rRNA; one was within residues 697 to 1163 of the 5' external transcribed spacer, and the other one was between nucleotides 664 and 1021 of the 18S rRNA sequence. E2 RNA was cross-linked to a region within residues 3282 to 3667 of the 28S rRNA sequence. E3 RNA was cross-linked to a sequence between positions 1021 and 1639 of the 18S rRNA sequence. Primer extension analysis located psoralen adducts in E1, E2, and E3 RNAs that were enriched in high-molecular-weight fractions of nucleolar RNA. Some of these psoralen adducts might be cross-links of E1, E2, and E3 RNAs to large nucleolar RNA. Antisense oligodeoxynucleotide-targeted RNase H digestion of nucleolar extracts revealed accessible segments in these three small RNAs. The accessible regions were within nucleotide positions 106 to 130 of E1 RNA, positions 24 to 48 and 42 to 66 of E2 RNA, and positions 7 to 16 and about 116 to 122 of E3 RNA. Some of the molecules of these small nucleolar RNAs sedimented as if associated with larger structures when both nondenatured RNA and a nucleolar extract were analyzed.

Effect of troglitazone on the desaturases in a rat model of insulin-resistance induced by a sucrose-rich diet.

A sucrose-rich diet generates time-dependent metabolic disorders similar to those found in diabetes type 2. After 8 month (mo) this diet evoked in the rat an increase of blood glucose, free fatty acids (FFA) and triacylycerides (TG) without insulin modification, an interruption of liver stearoyl-CoA desaturase-1 (SCD-1) mRNA and activity increase found at 6 mo, and an enhacement of Delta6 and Delta5 desaturase mRNA and Delta6 activity. We found that the administration of troglitazone (TRO), a peroxisome-proliferator-activated receptors gamma (PPAR-gamma) agonist, for 2 mo normalized plasma FFA, TG, and glucose without altering the insulinemia. It depressed liver SCD-1 mRNA in both control and sucrose-fed rats, decreasing the 18:1n-9/18:0 ratio in serum and liver lipids, and eliminated the increasing effect on mRNA and activity of Delta6 and Delta5 desaturases. These findings evidence again that desaturases are not affected through an insulin resistant effect evoked by the sucrose-rich diet and TRO recovers the altered metabolic plasma parameters as it corresponds to a PPAR-gamma agonist, but its effect on hepatic desaturases can not be attributed to a direct action on liver by PPAR-gamma, insulin, and even by an insulin sensitizing mechanism, suggesting it would be evoked indirectly through hepatic PPAR-alpha deactivation induced by the FFA decrease.

Detection and quantification of a very high density lipoprotein in different tissues of Triatoma infestans during the last nymphal and adult stages.

The presence of a very high density lipoprotein (VHDL), an hexameric protein, was explored in different tissues of Triatoma infestans throughout the last nymphal and adult stages, and in egg extracts by Western blot assays. The VHDL was always detected in both, hemolymph and fat body, during the above mentioned stages and it was also observed in the buffer soluble fraction of testis and egg homogenates. An enzyme-linked immunosorbent assay (ELISA) was used to measure the VHDL titer in these tissues. Hemolymph VHDL reaches a maximum value before the last molt, then it abruptly declines in males and females just after emergence, but during adult life it increases again. Fat body VHDL decreases slowly and continuously during the nymph growth reaching a minimum value prior to molting, and in the first week of adult life the values were even two-fold lower; then, it shows a different cycle of accumulation and depletion in males and females. In adult testis the VHDL undergoes a cycle similar to the one observed in male fat body. This protein increases progressively during embryonic development and, at the time of larval hatching it reaches its maximum value. The hexameric protein presents homologies in its N-terminal sequence with storage hexamerins of Diptera, Lepidoptera and Hymenoptera.

Purification and properties of a small lipid-binding protein from the hemolymph of Triatoma infestans.

A small lipid-binding protein (sLBP) was purified from the hemolymph of the blood-sucking bug Triatoma infestans. Its isolation involved size exclusion-high performance liquid chromatography (HPLC) followed by anion exchange chromatography-HPLC. The molecular weight of the protein, as determined by gel permeation chromatography, was 20 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved the protein into a single polypeptide with M(r) approximately equal to 16 kDa. The sLBP contains 6% lipids. Diacylglycerols represent the major lipid class, whereas phosphatidyl-choline, phosphatidyl-ethanolamine, free fatty acids and hydrocarbons were found in minor amounts. The amino acid composition indicated a high content of aspartic and glutamic acids and non-polar aliphatic amino acids. The N-terminal sequence did not resemble the sequence of any other previously reported insect hemolymph protein. Far-UV circular dichroism suggested that sLBP adopts a conformation rich in beta-sheet structure. The presence of this protein in hemolymph, fat body and unfertilized eggs was explored throughout the last nymphal and adult stages of the insect by Western blot assays. These assays indicated that sLBP is particularly abundant in hemolymph. A high concentration of sLBP was also detected in the fat body of the nymphs.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans.

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.

Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction.

Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.

Gene therapy for long-term restoration of circulating thymulin in thymectomized mice and rats.

Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.

Mosquito larvae soluble fractions induce DNA synthesis alterations on human mononuclear cells.

Mosquito larvae soluble fractions obtained by molecular exclusion chromatography altered the mitotic rate of several epithelial cell populations in hepatectomised mice, as well as the proliferation of human mononuclear cells (MNC), stimulating or inhibiting them depending on the fraction and dose applied. The effect was also thermolabile, suggesting a proteic nature of the compounds involved. Analysis of cell viability after culture indicated that the extract did not have lethal toxic effects. One fraction with a molecular weight ranging between 12-80 kDa caused only an inhibitory effect. In the present study, we performed further characterisation of this fraction by assaying the effect of new fractions obtained from this one, by the use of a column with a lower molecular weight exclusion range. Assays were performed on the proliferation of adult human MNCs. Our results showed that two out of four of the sub-fractions analysed, with a MW of about 70 and 17 kDa, caused a dose-dependent response, either inhibiting or stimulating MNC proliferation respectively.

Effect of phospholipids on the structure of Triatoma infestans lipophorin studied by fluorescence methods.

To study the role of phospholipids in the lipophorin structure, they were removed by phospholipase A2 treatment. Fluorescence lifetimes and accessibility to acrylamide quenching of different diphenylhexatrienyl derivatives, which were used as analogues of the different lipid components, indicate a surface localization of phospholipids and a distribution of diacylglycerols between the core and the surface, the surface fraction being increased by the phospholipase A2 treatment. The rotational behavior of these probes, studied by differential polarized phase fluorescence, indicates a high lipid order not only in the surface layer where phospholipids are located, but also in the core occupied by diacylglycerols and hydrocarbons. Phospholipid depletion increases the order only in the external region of the surface layer. Energy transfer from apolipoprotein tryptophan residues to several fluorescent acceptors indicates that phospholipid depletion produces a re-accommodation of the apoproteins. A decreased mobility of the water in the interfacial region is also produced by the phospholipase A2 treatment as it is reported by the fluorescence of 6-lauroyl-2-dimethylaminonaphthalene. This work shows that phospholipase A2 treatment of T. infestans lipophorin results in stable particles with an increased diacylglycerol to phospholipid ratio in the surface lipid layer. The modified particles are possibly stabilized by a conformational change in the apolipoproteins.

Lipid thermotropic transitions in Triatoma infestans lipophorin.

The structure and lipid thermotropic transitions of highly purified lipophorin of Triatoma infestans were examined by several techniques: steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), cis-parinaric acid (cis-PnA) and trans-parinaric acid (trans-PnA), light scattering fluorescence energy transfer between the lipophorin tryptophan residues and the bound chromophores, DPH, trans-parinaric acid cis-parinaric acid, gel electrophoresis, and gel filtration. Fluorescence polarization of PnAs and DPH revealed a reversible lipid thermotropic transition in intact lipophorin at about 20 degrees C and 18 degrees C, respectively. In lipophorin, lipid dispersion fluorescence polarization of DPH detected a lipid transition approximately at 20 degrees C, while trans-PnA showed a gel phase formation at a temperature below 30 degrees C. Similar experiments in which trans-PnA was incorporated into diacylglycerols and phospholipids extracted from the lipophorin revealed gel phase formation below 30 degrees C and 24 degrees C, respectively. Light scattering measurements showed that lipophorin particles aggregate irreversibly at 45 degrees C, increasing the molecular weight, as determined by gel filtration on Sephacryl S-300, from 740,000 to values larger than 1,500,000. The particle aggregation did not change the physical properties of the lipophorin studied by fluorescence polarization, indicating that the aggregation is apparently a non-denaturing process. Energy transfer between the lipophorin tryptophans and the bound chromophores cis-PnA, trans-PnA, and DPA revealed a different location of the fluorescent probes within the lipophorin. Temperature-dependence on the energy transfer efficiency for all probes confirmed a change in the ordering of the lipophorin lipids at 24 degrees C.

Biochemistry of the evolution of Triatoma infestans. XII. Biosynthesis and secretion of a very high density lipoprotein.

Biosynthetic processes related to the production of an insect hexamerin, very high density lipoprotein (VHDL), have been examined in the fat body of fifth-instar nymph and adult Triatoma infestans. Fat bodies were incubated in vitro with [3H]leucine and the incubation media were precipitated using a specific antiserum. The SDS-polyacrylamide gel electrophoresis followed by blotting on nitrocellulose showed that both larval and adult fat body secreted the VHDL subunit. Moreover, the radiolabel recovered in this subunit is indicative of the de novo synthesis. When the incubation medium was subjected to density gradient ultracentrifugation, a radiolabeled fraction was found at density 1.27 g/ml, value identical to the hemolymph circulating VHDL, indicating that the secreted apoprotein is combined with lipids. The SDS-polyacrylamide gel electrophoresis and immunoblotting of this fraction corroborated the presence of the VHDL-apoprotein. These results demonstrate that the fat body of T. infestans is able to synthesize the protein subunit which is associated to lipids as a lipoprotein particle that is released into the medium as VHDL.

Effects of diabetes and insulin on hepatic delta6 desaturase gene expression.

It has been recognized that rat liver microsomal Delta6 desaturation activity is defective in experimental diabetes, a fact that may be reverted by means of insulin treatment. In the present study, we used streptozotocin-induced diabetic rats in order to determine the regulatory role of insulin on the expression of hepatic Delta6 desaturase gene. The abundance of hepatic Delta6 desaturase mRNA in the diabetic rats is sevenfold lower than in the control. Insulin administration to diabetic rats induces Delta6 desaturase mRNA eightfold within 24 h. The effect of insulin on the Delta6 desaturase mRNA was inhibited 70% with dibutyryl-cAMP and theophylline administration and 90% by cycloheximide administration. Therefore, our data demonstrate that the activity of hepatic Delta6 desaturase in response to insulin is, at least in part, regulated by pretranslational events that require the synthesis of an unknown protein(s).

Partial characterization of mosquito larvae extract inducing dna synthesis alterations on human mononuclear cells.

A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12-80 kDa) showed only an inhibitory effect on cell proliferation.

Transport and utilization of free fatty acids in Triatoma infestans.

Triatoma infestans hemolymph has 0.31 mg/ml of free fatty acids and 2.8 mg/ml of diacylglycerols. Almost all the diacylglycerols are transported by lipophorin whereas free fatty acids are carried by lipophorin and a very high density lipoprotein. The binding of cis-parinaric acid to lipophorin was employed to specify the free fatty acid binding properties of lipophorin. Lipophorin has 10 binding sites of high affinity (3 x 10(7)) and approximately 45 binding sites of low affinity (1 x 10(6)). The relative rate of tissue incorporation of free fatty acids and diacylglycerols was measured by injecting insects with hemolymph previously labeled in both, free fatty acids and diacylglycerols. In this way, the half-life of the hemolymph free fatty acids was estimated to be about 4 min. Based on this result and taking into account the content of free fatty acids and diacylglycerols in hemolymph, the incorporation of free fatty acids, expressed in moles of fatty acids, seems to be 3.4 times higher than that of diacylglycerols. This finding can be applied to other insects.

Neuroendocrinology of aging: the potential of gene therapy as an interventive strategy.

OBJECTIVE: This paper reviews the current status of gene therapy in the neuroendocrine system and discusses the interventive potential of this methodology for neuroendocrine pathologies associated with aging. BACKGROUND AND RESULTS: A brief description is first presented of the viral-vector-based gene delivery systems being currently used in the neuroendocrine system, namely the adenoviral and herpetic (HSV1) vector systems. Next, an account of the neuroendocrine pathologies for which gene therapy approaches in animal models are being implemented is provided. This includes the treatment of experimental pituitary tumors by adenoviral-vector-mediated transfer of the suicide gene for the HSV-1 thymidine kinase. At the hypothalamic level, an adenovirus harboring the cDNA for arginine vasopressin has been used in Brattleboro rats to correct their diabetes insipidus. Next, the interventive potential of gene therapy for correcting age-associated neurodegenerative processes at neuroendocrine level is outlined. Finally, the role that emerging technologies may play in the development of future genetic therapies for aging is considered. CONCLUSION: Although effective implementation of gene therapy strategies still faces significant technical obstacles, these are likely to be progressively overcome as gene delivery systems are refined.

Three small nucleolar RNAs of unique nucleotide sequences.

Three small RNA species were detected in human cells, and their cDNAs were synthesized and cloned. These RNAs are nucleolar, are 207, 154, and 135 nucleotides long, and are named E1, E2, and E3, respectively, and their unique nucleotide sequences suggest that they may belong to an additional family of small nucleolar RNAs. The 5' ends of these three RNAs do not appear to have a trimethylguanosine cap or another type of cap. Apparent homologs of these three RNAs were detected in mouse, rabbit, and frog cells, suggesting their universal importance. They are housekeeping RNA species, since they are present in all rabbit tissues analyzed.

Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation.

Mosquito larvae crude extract have been found to alter the mitotic rate of several mouse epithelial cell populations such as enterocytes and tongue keratinocytes. Also, the dialysed fraction inhibits hepatocyte proliferation in hepatectomized males. These experiments suggested an inhibitory effect on the G1/S interphase. Consequently, we suggested the presence of some molecule or molecules related to the TGF-beta superfamily. In the present paper, we have assayed the crude extract on human mononuclear cells and the dialysed fraction of the extract on tongue keratinocyte proliferation. Furthermore, different protein fractions obtained using a molecular exclusion chromatographic column were assayed on hepatocyte proliferation of hepatectomized mice. Three groups of proteins have been isolated. Results show a dose-dependent effect of crude extract on mononuclear cell proliferation and the dialysed extract caused an inhibitory effect on tongue keratinocyte proliferation. With regard to the hepatocyte mitotic rate, an inhibitory effect appeared only in animals receiving the fraction with lower molecular weight. These results suggest the presence in mosquito larvae of some peptidic molecule or molecules resembling the activity of members of the TGF-beta superfamily.

Purification and properties of the very high density lipoprotein from the hemolymph of adult Triatoma infestans.

The very high density lipoprotein (VHDL) of Triatoma infestans hemolymph from adult males has been isolated and purified by two-step density gradient ultracentrifugation. It appears to be homogeneous as judged by native polyacrylamide gel electrophoresis. The content of VHDL in hemolymph was estimated to be 8 mg protein/ml. The purified protein has a molecular weight (Mr) of 450,000, is composed of six subunits of Mr approximately equal to 77,000, and possesses a high content of aromatic amino acids. This protein is glycosylated and contains 3% of lipids by weight with a remarkable amount of free fatty acids (25% of total lipids). The T. infestans VHDL has a different lipid and amino acid composition from lipophorin. The lipid composition and the spectroscopic studies using cis-parinaric acid indicated a high fatty acid binding affinity. It has nine binding sites per mol of VHDL. Competence studies revealed that VHDL has its highest affinity for the binding of palmitic acid followed by stearic and arachidonic acids.

Deletion analysis of the enolase gene (enoA) promoter from the filamentous fungus Aspegillus oryzae.

The enolase gene (enoA) is one of the most strongly expressed genes in Aspergillus oryzae. To elucidate the transcription regulatory element for this strong expression and the process of glucose induction, the transcription activity of a series of truncated enoA promoters was measured by using the Escherichia coli uidA gene as a reporter. Deletion of a 104-bp region located -224 nt to -121 nt upstream of the translation initiation site caused both a drastic decrease in the beta-glucuronidase (GUS) activity and a loss of glucose induction. Northern blot analysis confirmed that the decrease in GUS activity was achieved at the transcriptional level. In addition, electrophoretic gel mobility shift assays indicated that the 104-bp region contained a 15-bp element, to which one or more A. oryzae cellular factors specifically bind. These results suggest that the 15-bp element between -195 nt and -181 nt includes the sequence essential for the transcription regulation of the A. oryzae enoA gene.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.