D-Met2,Pro5] enkephalin [N1.5-beta-D-glucopyranosyl] amide: a glycosylpeptide with high antinociceptive activity

Different synthetic strategies have been attempted for the synthesis of a glycosylpeptide resulting from the covalent bonding of a sugar residue to the C-terminal carboxyl group of an enkephalin related pentapeptide. The final structure is: Tyr-D-Met-Gly-Phe-Pro [N1.5-beta-D-glucopyranosyl] amide. The in vitro potency on the GPI test of this analogue was IC50 = 64.0 nM. However, its antinociceptive activity by tail immersion tests, after intraperitoneal administration, was 2000 and 200 times higher than morphine in rats and mice, respectively.     

Phosphorylation of myosin light chain from adrenomedullary chromaffin cells in culture.

The myosin-light-chain (MLC) phosphorylation accompanying catecholamine release in chromaffin cells was investigated with the objective of assessing the possible role of this contractile protein in catecholamine secretion. The electrophoretic characteristics of adrenomedullary MLC were determined by immunochemical techniques using two different specific antibodies. The identified 22 kDa phosphoprotein was mainly present in the cytosol, as demonstrated by ultracentrifugation and immunocytochemical analysis. A part of this protein was located on, or close to, the plasma membrane. Cell stimulation by secretagogues resulted in a Ca2(+)-dependent 32P incorporation into MLC, the time course of this process being related to catecholamine release. These findings were supported by a two-dimensional gel-electrophoretic analysis by which means this protein was resolved into two acidic forms. A role for Ca2(+)-calmodulin and Ca2(+)-phospholipid kinases in adrenomedullary MLC phosphorylation is reported. The results obtained suggest a regulatory role for such a protein in the underlying exocytotic event.     

Pineal transduction. Adrenergic----cyclic AMP-dependent phosphorylation of cytoplasmic 33-kDa protein (MEKA) which binds beta gamma-complex of transducin

Adrenergic regulation of phosphorylation of pineal proteins was studied. Norepinephrine treatment of intact pinealocytes incubated with 32Pi enhanced phosphorylation of a 33-kDa phosphoprotein (33PP). The effect of NE was rapid, sustained, and appeared to be mediated by a beta-adrenergic----cyclic AMP mechanism. Studies using broken cell preparations revealed that 33PP was phosphorylated by cyclic AMP-dependent protein kinase (PKA). It was also possible to demonstrate PKA-dependent phosphorylation of the 33-kDa protein in cytosol from rat retina and in cow and sheep pineal glands. Two-dimensional polyacrylamide gel electrophoresis revealed that 33PP is acidic (pI congruent to 4.5), appears to exist as two isoforms with slightly different charge, and has the same mobility as the retinal 33-kDa PKA substrate. Immunological analysis indicated 33PP in both tissues is a previously reported 33-kDa protein (MEKA); this protein is a PKA substrate which has been reported to form a cytoplasmic complex with the beta gamma complex of transducin. Consistent with this, it was possible to identify the beta-subunit in pineal cytoplasm and in the same congruent to 70-kDa gel permeation fraction which contained the 33-kDa protein identified as MEKA. Thus, it appears possible that MEKA is present in pineal cytoplasm in a 70-kDa complex with G beta gamma, as is the case in retina. The finding of MEKA in the pineal makes it the latest addition to a family of retinal/pineal proteins which are thought to have evolved from a common ancestral photochemical transduction system.     

The effect of the structure of branched polypeptide carrier on intracellular delivery of daunomycin

The conjugate of acid labile cis-aconityl-daunomycin (cAD) with branched chain polypeptide, poly[Lys(Glui-DL-Alam)] (EAK) was very effective against L1210 leukemia in mice. However, Dau attached to a polycationic polypeptide, poly[Lys(Seri-DL-Alam)] (SAK) exhibited no in vivo antitumor effect. In order to understand this difference we have performed comparative in vitro studies to dissect properties related to interaction with the whole body (e.g., biodistribution) from those present at cellular or even molecular level. We report here (a) the kinetics of acid-induced Dau liberation, (b) interaction with DPPC phospholipid bilayer, (c) in vitro cytotoxic effect on different tumor cells, and (d) intracellular distribution in HL-60 cells of polycationic (cAD-SAK) and amphoteic (cAD-EAK) conjugates. Fluorescence properties of the two conjugates are also reported. Our findings demonstrate that the kinetics of the drug release, intracellular distribution and in vitro cytotoxic effect are rather similar, while the effect on DPPC phospholipid bilayer and fluorescence properties of the two conjugates are not the same. We also found that the in vitro cytotoxicity is cell line dependent. These observations suggest that the structure of the polypeptide carrier could have marked influence on drug uptake related events.     

Optimization study of doxorubicin liposomal preparations coated with laminin fragments

Immunoliposomes, coated with two peptide sequences and loaded with doxorubicin, were prepared. The influence of different parameters in the sequential steps of liposomal preparations was studied as, for instance, lipid composition, size reduction methods, elimination of non-entrapped drug, and peptide coating sequence. Results were evaluated, such as entrapment efficiency, phospholipid/drug and phospholipid/peptide relationship, and size of final preparations. Effective size reduction was only achieved through probe sonication and the presence of peptides on the surface of liposomes, which does not modify, significantly, the final phospholipid/drug relationship, related to the initial values; however, they promoted a slight increase in the size of final preparations. Dialysis was the most suitable method to wash liposomes from reactants, drug and peptides, as well as being the cleanest process to avoid microbial contamination without significant dilution. Peptide coating yields were similar for liposomal compositions presenting free carboxyl groups on the surface. As determined by other authors, the presence of polyethylene glycol monomethoxy chains on the surface reduces the reactivity of NPGE carboxylic groups.     

The evolutionary history of Drosophila buzzatii XXVII

The correlation between body size and longevity was tested in an Argentinian natural population of Drosophila buzzatii. Mean thorax length of flies newly emerging from rotting cladodes of Opuntia vulgaris was significantly smaller than that of two samples of flies caught at baits. The present results which might be interpreted as directional selection for longevity favoring larger flies are in agreement with previous results achieved in a Spanish natural population of D. buzzatii. Flies emerging from different substrates showed significant differences in thorax length, suggesting that an important fraction of phenotypic variance can be attributed to environmental variability. However, laboratory and field work in different populations of D. buzzatii showed a significant genetic component for thorax length variation.     

Nutrient toxicity in pancreatic β-cell dysfunction

Nutrients, such as glucose and fatty acids, have a dual effect on pancreatic beta-cell function. Acute administration of high glucose concentrations to pancreatic beta-cells stimulates insulin secretion. In addition, short term exposure of this cell type to dietary fatty acids potentiates glucose-induced insulin release. On the other hand, long-term exposure to these nutrients causes impaired insulin secretion, characterized by elevated exocytosis at low concentrations of glucose and no response when glucose increases in the extracellular medium. In addition, other phenotypic changes are observed in these conditions. One major step in linking these phenotypic changes to the diabetic pathology has been the recognition of both glucose and fatty acids as key modulators of beta-cell gene expression. This could explain the adaptative response of the cell to sustained nutrient concentration. Once this phase is exhausted, the beta-cell becomes progressively unresponsive to glucose and this alteration is accompanied by the irreversible induction of apoptotic programs. The aim of this review is to present actual data concerning the development of the toxicity to the main nutrients glucose and fatty acids in the pancreatic beta-cell and to find a possible link to the development of type 2 diabetes.     

Differences in secondary structure of HAV-synthetic peptides induced by the sequential order of T- and B-cell epitopes

The present study was undertaken to examine the structural features of two peptide constructs designed on the basis of linear combination of B and T-cell epitopes in different orientations (BT and TB) that may be important to explain the differences in the elicited antihepatitis A virus immune response and in the interaction with biological model membranes. A CD study was carried out and the corresponding quantitative analysis of the experimental data was done using deconvolution computer programs. Moreover, fluorescence experiments were performed to analyze differences in the fluorescence emission spectra of both molecules. The main conformational difference by CD studies was obtained working in aqueous medium. Although the TB sequence adopted a preferably random coil structure, the BT peptide was best fitted with beta-type structures. These results are further supported by fluorescence studies. These findings have relevance for the design of synthetic immunopeptides.     

Development of diagnostic markers for selection of the subacid trait in peach

Peaches with low acidity are preferred in the market and this trait is usually selected in commercial breeding programs. A major gene (D/d) has been described for this character located on linkage group 5 of peach, where the low acid character is determined by the dominant D allele. In this paper, we analyze a collection of 231 varieties and 542 offspring to identify diagnostic markers for this character. The CPPCT040 single sequence repeat (SSR) is known to be tightly linked to D. We found that one of its alleles (193) is diagnostic for the subacid character and identified with high probability individuals with low acidity (titratable acidity <5.5 mg/l). The region around CPPCT040 was explored using 13 DNA fragments for a total of 5,297 bp, covering a length of 70.4 kbp of the peach genome. The sequenced fragments detected 19 single nucleotide polymorphisms (SNPs) and five indels. All subacid individuals shared a large haplotype (>24 kb) around CPPCT040, a region with higher than average SNPs between acid and subacid varieties. The CPPCT040 marker plus one of the SNPs identified (DH875) were used to genotype a collection of 542 seedlings, from different crosses expected to segregate for this character, which were phenotyped by tasting the fruit in the field. Data provided by both markers were always consistent and only 24 plants (4 %) did not fit the expectations. These markers and others that can be obtained from the haplotype identified can be readily used for marker-assisted selection in peach breeding.